CA 02307010 2000-OS-19
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CA 02307010 2000-OS-19
TITLE OF THE INVENTION
HIGHLY CONSERVED GENES AN THEIR USE TO GENERATE SPECIES-
SPECIFIC, GENUS-SPECIFIC, FAMILY-SPECIFIC, GROUP-SPECIFIC AND
UNIVERSAL NUCLEIC ACID PROBES AND AMPLIFICATION PRIMERS TO
RAPIDLY DETECT AND IDENTIFY BACTERIAL, FUNGAL AND PARASITICAL
PATHOGENS FROM CLINICAL SPECIMENS FOR DIAGNOSIS
1o BACKGROUND OF THE INVENTION
Classical methods for the identification of bacteria, fungi, and parasites
Bacteria and fungi are classically identified by their ability to utilize
different
substrates as a source of carbon and nitrogen through the use of biochemical
tests
such as the API20ETM system (bioMerieux). For susceptibility testing, clinical
microbiology laboratories use methods including disk diffusion, agar dilution
and
broth microdilution. Although identifications based on biochemical tests and
antibacterial susceptibility tests are cost-effective, at least two days are
required to
obtain preliminary results due to the necessity of two successive overnight
incubations to identify the bacteria from clinical specimens as well as to
determine
their susceptibility to antimicrobial agents. There are some commercially
available
automated systems (i.e. the MicroScanTM system from Dade Diagnostics Corp. and
the VitekTM system from bioMerieux) which use sophisticated and expensive
apparatus for faster microbial identification and susceptibility testing
(Stager and
Davis, 1992, Clin. Microbiol. Rev. 5:302-327). These systems require shorter
incubation periods, thereby allowing most bacterial identifications and
susceptibility
testing to be performed in less than 6 hours. Nevertheless, these faster
systems always
require the primary isolation of the bacteria or fungi as a pure culture, a
process
which takes at least 18 hours for a pure culture or 2 days for a mixed
culture. So, the
shortest time from sample reception to identification of the pathogen is
around 24
hours. Moreover, fungi other than yeasts are often difficult or very slow to
cultivate
from clinical specimens. Identification must rely on labor-intensive
techniques such
as direct microscopic examination of the specimens and by direct and/or
indirect
immunological assays. Cultivation of most parasites is impractical in the
clinical
laboratory. Hence, microscopic examination of the specimen, a few
immunological
tests and clinical symptoms are often the only methods used for an
identification that
frequently remains presumptive.
The fastest bacterial identification system, the autoSCAN-Walk-AwayTM system
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CA 02307010 2000-OS-19
(Dade Diagnostics Corp.) identifies both gram-negative and gram-positive
bacterial
species from standardized inoculum in as little as 2 hours and gives
susceptibility
patterns to most antibiotics in 5.5 hours. However, this system has a
particularly high
percentage (i.e. 3.3 to 40.5%) of non-conclusive identifications with
bacterial species
other than Enterobacteriaceae (Croize J., 1995, Lett. Infectiol. 10:109-113;
York et
aL., 1992, J. Clin. Microbiol. 30:2903-2910). For Enterobacteriaceae, the
percentage
of non-conclusive identifications was 2.7 to 11.4%.
A wide variety of bacteria and fungi are routinely isolated and identified
from
clinical specimens in microbiology laboratories. Tables 1 and 2 give the
incidence for
the most commonly isolated bacterial and fungal pathogens from various types
of
clinical specimens. These pathogens are the main organisms associated with
nosocomial and community-acquired human infections and are therefore
considered
the most clinically important.
Clinical specimens tested in clinical microbiology laboratories
Most clinical specimens received in clinical microbiology laboratories are
urine
and blood samples. At the microbiology laboratory of the Centre Hospitalier de
1'Universite Laval (CHUL), urine and blood account for approximately 55% and
30%
of the specimens received, respectively (Table 3). The remaining 15% of
clinical
specimens comprise various biological fluids including sputum, pus,
cerebrospinal
fluid, synovial fluid, and others (Table 3). Infections of the urinary tract,
the
respiratory tract and the bloodstream are usually of bacterial etiology and
require
antimicrobial therapy. In fact, all clinical samples received in the clinical
microbiology laboratory are tested routinely for the identification of
bacteria and
susceptibility testing.
Conventional pathogen identification from clinical specimens
Urine specimens
The search for pathogens in urine specimens is so preponderant in the routine
microbiology laboratory that a myriad of tests have been developed. However,
the
gold standard remains the classical semi-quantitative plate culture method in
which 1
~,L of urine is streaked on plates and incubated for 18-24 hours. Colonies are
then
counted to determine the total number of colony forming units (CFU) per liter
of
urine. A bacterial urinary tract infection (UTI) is normally associated with a
bacterial
count of 107 CFU/L or more in urine. However, infections with less than 107
CFU/L
in urine are possible, particularly in patients with a high incidence of
diseases or
those catheterized (Stark and Maki, 1984, N. Engl. J. Med. 311:560-564).
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Importantly, approximately 80% of urine specimens tested in clinical
microbiology
laboratories are considered negative (i.e. bacterial count of less than 10~
CFU/L;
Table 3). Urine specimens found positive by culture are further characterized
using
standard biochemical tests to identify the bacterial pathogen and are also
tested for
susceptibility to antibiotics. The biochemical and susceptibility testing
normally
require 18-24 hours of incubation.
Accurate and rapid urine screening methods for bacterial pathogens would allow
a faster identification of negative specimens and a more efficient treatment
and care
management of patients. Several rapid identification methods (UriscreenTM,
UTIscreenTM, Flash TrackTM DNA probes and others) have been compared to slower
standard biochemical methods, which are based on culture of the bacterial
pathogens.
Although much faster, these rapid tests showed low sensitivities and poor
specificities as well as a high number of false negative and false positive
results
(Koening et al., 1992, J. Clin. Microbiol. 30:342-345; Pezzlo et al., 1992, J.
Clin.
Microbio1.30:640-684).
Blood specimens
The blood specimens received in the microbiology laboratory are always
submitted for culture. Blood culture systems may be manual, semi-automated or
completely automated. The BACTECTMSystem (from Becton Dickinson) and the
BacTAlertTM system (from Organon Teknika Corporation) are the two most widely
used automated blood culture systems. These systems incubate blood culture
bottles
under optimal conditions for bacterial growth. Bacterial growth is monitored
continuously to detect early positives by using highly sensitive bacterial
growth
detectors. Once growth is detected, a Gram stain is performed directly from
the blood
culture and then used to inoculate nutrient agar plates. Subsequently,
bacterial
identification and susceptibility testing are carried out from isolated
bacterial colonies
with automated systems as described previously. The bottles are normally
reported as
negative if no growth is detected after an incubation of 6 to 7 days.
Normally, the
vast majority of blood cultures are reported negative. For example, the
percentage of
negative blood cultures at the microbiology laboratory of the CHUL for the
period
February 1994-January 1995 was 93.1 % (Table 3).
Other clinical samples
Upon receipt by the clinical microbiology laboratory, all body fluids other
than
blood and urine that are from normally sterile sites (i.e. cerebrospinal,
synovial,
pleural, pericardial and others) are processed for direct microscopic
examination and
subsequent culture. Again, most clinical samples are negative for culture
(Table 3). In
these normally sterile sites, a test for the universal detection of bacteria,
fungi and
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CA 02307010 2000-OS-19
parasites would be very useful.
Regarding clinical specimens which are not from sterile sites such as sputum
or
stool specimens, the laboratory diagnosis by culture is more problematic
because of
the contamination by the normal flora. The bacterial or fungal pathogens
potentially
associated with the infection are grown and separated from the colonizing
microbes
using selective methods and then identified as described previously. Of
course, the
universal detection of bacteria would not be useful for the diagnosis of
bacterial
infections at these non-sterile sites. On the other hand, DNA-based assays for
species
or genus or family or group detection and identification as well as for the
detection of
antibiotic resistance genes from these specimens would be very useful and
would
offer several advantages over classical identification and susceptibility
testing
methods.
DNA-based assays with any specimen
There is an obvious need for rapid and accurate diagnostic tests for the
detection and identification of bacteria, fungi and parasites directly from
clinical
specimens. DNA-based technologies are rapid and accurate and offer a great
potential
to improve the diagnosis of infectious diseases (Persing et ad., 1993,
Diagnostic
Molecular Microbiology: Principles and Applications, American Society for
Microbiology, Washington, D.C.; Bergeron and Ouellette, 1995, Infection 23:69-
72;
Bergeron and Ouellette, 1998, J Clin Microbiol. 36:2169-72). The DNA probes
and
amplification primers which are objects of the present invention are
applicable for the
detection and identification of bacteria, fungi, and parasites directly from
any clinical
specimen such as blood cultures, blood, urine, sputum, cerebrospinal fluid,
pus,
genital and gastro-intestinal tracts, skin or any other type of specimens
(Table 3). The
DNA-based tests proposed in this invention are superior in terms of both
rapidity and
accuracy to standard biochemical methods currently used for routine diagnosis
from
any clinical specimens in microbiology laboratories. Since these tests can be
performed in one hour or less, they provide the clinician with new diagnostic
tools
which should contribute to a better management of patients with infectious
diseases.
Specimens from sources other than humans (e.g. other primates, birds, plants,
mammals, farm animals, livestock, food products, environment such as water or
soil,
and others) may also be tested with these assays.
A high percentage of culture negative specimens
Among all the clinical specimens received for routine diagnosis, approximately
80% of urine specimens and even more (around 95%) for other types of normally
sterile clinical specimens are negative for the presence of bacterial
pathogens (Table
3). It would also be desirable, in addition to identify bacteria at the
species or genus
or family or group level in a given specimen, to screen out the high
proportion of
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CA 02307010 2000-OS-19
negative clinical specimens with a test detecting the presence of any
bacterium (i.e.
universal bacterial detection). Such a screening test may be based on DNA
amplification by PCR of a highly conserved genetic target found in all
bacteria.
Specimens negative for bacteria would not be amplified by this assay. On the
other
hand, those that are positive for any bacterium would give a positive
amplification
signal with this assay. Similarly, highly conserved genes of fungi and
parasites could
serve not only to identify particular species or genus or family or group but
also to
detect the presence of any fungi or parasite in the specimen.
1o Towards the development of rapid DNA-based diagnostic tests
A rapid diagnostic test should have a significant impact on the management of
infections. DNA probe and DNA amplification technologies offer several
advantages
over conventional methods for the identification of pathogens and antibiotic
resistance genes from clinical samples (Persing et al., 1993, Diagnostic
Molecular
Microbiology: Principles and Applications, American Society for Microbiology,
Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-based Diagnostics in
Infectious Disease, Blackwell Scientific Publications, Boston, MA). There is
no need
for culture of the pathogens, hence the organisms can be detected directly
from
clinical samples, thereby reducing the time associated with the isolation and
identification of pathogens. Furthermore, DNA-based assays are more accurate
for
microbial identification than currently used phenotypic identification systems
which
are based on biochemical tests and/or microscopic examination. Commercially
available DNA-based technologies are currently used in clinical microbiology
laboratories, mainly for the detection and identification of fastidious
bacterial
pathogens such as Mycobacterium tuberculosis, Chlamydia trachomatis, Neisseria
gonorrhoeae as well as for the detection of a variety of viruses (Podzorski
and
Persing, Molecular detection and identification of microorganisms, In: P.
Murray et
al., 1995, Manual of Clinical Microbiology, ASM press, Washington D.C.). There
are
also other commercially available DNA-based assays which are used for culture
confirmation assays.
Others have developed DNA-based tests for the detection and identification of
bacterial pathogens which are objects of the present invention for example:
Staphylococcus sp. (US patent serial no. 5,437,978), Neisseria sp. (US patent
serial
no. 5,162,199 and European patent serial no. 0,337,896,131) and Listeria
monocytogenes (US patent serial nos. 5,389,513 and 5,089,386). However, the
diagnostic tests described in these patents are based either on rRNA genes or
on
genetic targets different from those described in the present invention. To
our
knowledge there are only three patents published by others mentioning the use
of any
of the three targets described in the present invention for diagnostic
purposes (PCT
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CA 02307010 2000-OS-19
international publication number W092/03455, European patent publication
number
0 133 671 B1, and European patent publication number 0 133 288 A2). W092/03455
is focused on the inhibition of Candida species for therapeutic purposes. It
describes
antisense oligonucleotide probes hybridizing to Candida messenger RNA. Two of
the
numerous mRNA proposed as target are coding for translation elongation factor
1
(tef 1 ) and the beta subunit of ATPase. DNA amplification or hybrization are
not
under the scope of their invention and although diagnostic use is briefly
mentioned in
the body of the application, no specific claim is made regarding diagnostics.
In the
main body of the text, EP 0 133 671 B 1 describes the use of bacterial tuf
sequence for
diagnostics based on hybridization with bacterial RNA. To hybridize RNA, an
oligonucleotide probe must be antisense. DNA amplification techniques require
the
use of both sense and antisense primers. Hence, claim number one from EP 0 133
671
B 1 precludes PCR or other DNA-based amplification techniques. Furthermore, EP
0
133 671 B 1 makes no specific claim on the use of tuf sequences for
diagnostics; only
ribosomal RNA sequences are claimed. Patent EP 0 133 288 A2 describes and
claims
the use of bacterial tuf sequence for diagnostics based on hybridization of a
tuf probe
with bacterial DNA. DNA amplification is not in the scope of EP 0 133 288 A2.
Nowhere it is mentioned that multiple tuf probes could be used simultaneously.
The
sensitivity of the tuf hybrizations reported are, at 1x106 bacteria or 1-100
ng of DNA,
much lower than those achievable by nucleic acid amplification technologies.
Although there are phenotypic identification methods which have been used for
more than 125 years in clinical microbiology laboratories, these methods do
not
provide information fast enough to be useful in the initial management of
patients.
There is a need to increase the speed of the diagnosis of commonly encountered
bacterial, fungal and parasitical infections. Besides being much faster, DNA-
based
diagnostic tests are more accurate than standard biochemical tests presently
used for
diagnosis because the microbial genotype (e.g. DNA level) is more stable than
the
phenotype (e.g. physiologic level).
Knowledge of the genomic sequences of bacterial, fungal and parasitical
species continuously increases as testified by the number of sequences
available from
public databases such as GenBank. From the sequences readily available from
those
public databases, there is no indication therefrom as to their potential for
diagnostic
purposes. For determining good candidates for diagnostic purposes, one could
select
sequences for DNA-based assays for (i) the species-specific detection and
identification of commonly encountered bacterial, fungal and parasitical
pathogens,
(ii) the genus-specific detection and identification of commonly encountered
bacterial, fungal or parasitical pathogens, (iii) the family-specific
detection and
identification of commonly encountered bacterial, fungal or parasitical
pathogens,
(iv) the group-specific detection and identification of commonly encountered
bacterial, fungal or parasitical pathogens, (v) the universal detection of
bacterial,
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fungal or parasitical pathogens, and/or (vi) the specific detection and
identification of
antibiotic resistance genes, and/or (vii) the specific detection and
identification of
bacterial toxin genes. All of the above types of DNA-based assays may be
performed
directly from any type of clinical specimens or from a microbial culture.
In our co-pending U.S. patent 6,001,564 and W098/20157 patent application,
we described DNA sequences suitable for (i) the species-specific detection and
identification of clinically important bacterial pathogens, (ii) the universal
detection
of bacteria, and (iii) the detection of antibiotic resistance genes.
The latter co-pending application described proprietary tuf DNA sequences as
well as tuf sequences selected from public databases (in both cases, fragments
of at
least 100 base pairs), as well as oligonucleotide probes and amplification
primers
derived from these sequences. All the nucleic acid sequences described in that
patent
application can enter in the composition of diagnostic kits or product and
methods
capable of a) detecting the presence of bacteria, fungi and parasites b)
detecting
specifically at the species, genus, family or group levels, the presence of
bacteria,
fungi and parasites and antibiotic resistance genes associated with these
pathogens.
However, these methods and kits need to be improved, since the ideal kit and
method
should be capable of diagnosing close to 100% of microbial pathogens and
associated
antibiotic resistance genes and toxins genes. For example, infections caused
by
Enterococcus faecium have become a clinical problem because of its resistance
to
many antibiotics. Both the detection of these bacteria and the evaluation of
their
resistance profiles are desirable. Besides that, novel DNA sequences (probes
and
primers) capable of recognizing the same and other microbial pathogens or the
same
and additional antibiotic resistance genes are also desirable to aim at
detecting more
target genes and complement our earlier patent applications.
The present invention improves the co-pending application by disclosing new
proprietary tuf sequences as well as describing new ways to obtain tuf
sequences. In
addition we disclose new proprietary atpD and recA sequences. In addition, new
uses
of tuf, atpD and recA DNA sequences selected from public databases (Table 11 )
are
disclosed.
Highly conserved genes for identification and diagnostics
Highly conserved genes are useful for identification of microorganisms. For
bacteria, the most studied genes for identification of microorganisms are the
universally conserved ribosomal RNA genes (rRNA). Among those, the principal
targets used for identification purposes are the small subunit (SSU) ribosomal
16S
rRNA genes (in prokaryotes) and 18S rRNA genes (in eukaryotes) (Relman and
Persing, Genotyping Methods for Microbial Identification, In: D.H. Persing,
1996,
PCR Protocols for Emerging Infectious Diseases, ASM Press, Washington D.C.).
The
rRNA genes are also the most commonly used targets for universal
identification of
bacteria (Chen et al., 1988, FEMS Microbiol. Lett. 57:19-24; McCabe et al.,
1999,
Mol. Genet. Metabol. 66:205-211 ) and fungi (Van Burik et al., 1998, J. Clin.
Microbiol. 36:1169-1175).
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However, it may be difficult to discriminate between closely related species
when using primers derived from the 16S rRNA. In some instances, 16S rRNA
sequence identity may not be sufficient to guarantee species identity (Fox et
al.,
1992, Int. J. Syst. Bacteriol. 42:166-170) and it has been shown that inter-
operon
sequence variation as well as strain to strain variation could undermine the
application of 16S rRNA for identification purposes (Clayton et al., 1995,
Int. J. Syst.
Bacteriol. 45:595-599).
STATEMENT OF THE INVENTION
It is an object of the present invention to provide a specific, ubiquitous and
sensitive method using probes and/or amplification primers for determining the
presence andlor amount of nucleic acids:
- from any bacterial, fungal or parasitical species
in any sample suspected of containing said nucleic acids, and optionally,
- from specific microbial species or genera selected from the group consisting
of the species or genera listed in Table 4
- from an antibiotic resistance gene selected from the group consisting of the
genes listed in Table 5, and optionally,
- from a toxin gene selected from the group consisting of the genes listed in
Table 6,
wherein each of said nucleic acids or a variant or part thereof comprises a
selected target region hybridizable with said probe or primers;
said method comprising the steps of contacting said sample with said probes or
primers and detecting the presence and/or amount of hybridized probes or
amplified
products as an indication of the presence andlor amount of said any microbial
species, specific microbial species or genus or family or group and antibiotic
resistance gene andlor toxin gene.
In a specific embodiment, a similar method directed to each specific microbial
species or genus or family or group detection and identification, antibiotic
resistance
genes detection, toxin genes detection, and universal bacterial detection,
separately, is
provided.
In a more specific embodiment, the method makes use of DNA fragments from
conserved genes (proprietary sequences and sequences obtained from public
databases), selected for their capacity to sensitively, specifically and
ubiquitously
detect the targeted bacterial, fungal or parasitical nucleic acids.
In a particularly preferred embodiment, oligonucleotides of at least 12
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CA 02307010 2000-OS-19
nucleotides in length have been derived from the longer DNA fragments, and are
used in the present method as probes or amplification primers.
In another particularly preferred embodiment, oligonucleotides primers and
probes of at least 12 nucleotides in length are designed for their specificity
and
ubiquity based upon analysis of our databases of tuf, atpD and recA sequences.
These
databases are generated using both proprietary and public sequence
information.
Altogether, these databases form a sequence repertory useful for the design of
primers and probes for the detection and identification of bacterial, fungal
and
parasitical microorganisms. The repertory can also be subdivided into
subrepertories
l0 for analysis leading to the design of primers and probes.
The tuf, atpD and recA sequences databases as a product to assist the design
of
oligonucleotides primers and probes for the detection and identification of
bacterial,
fungal and parasitical microorganisms are also an object of this invention.
The proprietary oligonucleotides (probes and primers) are also another object
of the invention.
Diagnostic kits comprising probes or amplification primers for the detection
of
a microbial species or genus or family or group selected from the following
list
consisting of Bordetella sp., Candida albicans, Candida dubliniensis, Candida
sp.,
Chlamydia pneumoniae, Chlamydia trachomatis, Clostridium sp., Corynebacterium
sp., Cryptosporidium parvum, Entamoeba sp., Enterobacteriaceae group,
Enterococcus casseliflavus-flavescens-gallinarum group, Enterococcus faecalis,
Enterococcus faecium, Enterococcus gallinarum, Enterococcus sp., Escherichia
coli,
Giardia sp., Haemophilus influenzae, Trypanosomatidae family, Leishmania sp.,
Mycobacteriaceae family, Neisseria gonorrhoeae, platelets contaminants group
(see
Table 14), Pseudomonads group, Staphylococcus aureus, Staphylococcus
epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis,
Staphylococcus
saprophyticus, Staphylococcus sp., Streptococcus agalactiae, Streptococcus
sp.,
Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma sp., are also objects of
the
present invention.
Diagnostic kits further comprising probes or amplification primers for the
detection of an antibiotic resistance gene selected from the group listed in
Table 5 are
also objects of this invention.
Diagnostic kits further comprising probes or amplification primers for the
detection of a toxin gene selected from the group listed in Table 6 are also
objects of
this invention.
Diagnostic kits further comprising probes or amplification primers for the
detection of any bacterial, fungal or parasitical species, comprising or not
comprising
those for the detection of the specific microbial species or genus or family
or group
listed above, and further comprising or not comprising probes and primers for
the
antibiotic resistance genes listed above, and further comprising or not
comprising
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CA 02307010 2000-OS-19
probes and primers for the toxin genes listed above are also objects of this
invention.
In a preferred embodiment, such a kit allows for the separate or the
simultaneous detection and identification of the above-listed microbial
species or
genus or family or group, antibiotic resistance genes, toxin genes and for the
detection of any microorganism (bacteria, fungi or parasite).
In the above methods and kits, amplification reactions may include but are not
restricted to: a) polymerase chain reaction (PCR), b) ligase chain reaction
(LCR), c)
nucleic acid sequence-based amplification (NASBA), d) self-sustained sequence
replication (3SR), e) strand displacement amplification (SDA), f) branched DNA
l0 signal amplification (bDNA), g) transcription-mediated amplification (TMA),
h)
cycling probe technology (CPT), i) nested PCR, j) multiplex PCR, k) solid
phase
amplification (SPA), 1) nuclease dependent signal amplification (NDSA), m)
rolling
circle amplification technology (RCAT).
In the above methods and kits, detection of the nucleic acids of target genes
may include real-time or post-amplification technologies. These detection
technologies can include, but are not limited to, fluorescence resonance
energy
transfer (FRET)-based methods such as adjacent hybridization to FRET probes
(including probe-probe and probe-primer methods), Taqman, molecular beacons
and
Scorpions. Other detection methods include target genes nucleic acids
detection via
immunological methods, solid phase hybridization methods on filters, chips or
any
other solid support, whether the hybridization is monitored by fluorescence,
chemiluminescence, potentiometry, mass spectrometry, plasmon resonance,
polarimetry, or colorimetry. Sequencing, including sequencing by dideoxy
termination or sequencing by hybridization, is another possible method to
detect and
identify the nucleic acids of target genes.
In a preferred embodiment, a PCR protocol is used for nucleic acid
amplification.
In a particularly preferred embodiment, a PCR protocol is provided,
comprising, an initial denaturation step of 1-3 minutes at 95 °C,
followed by an
amplification cycle including a denaturation step of one second at 95
°C and an
annealing step of 30 seconds at 45-65 °C, without any time allowed
specifically for
the elongation step. This PCR protocol has been standardized to be suitable
for PCR
reactions with most selected primer pairs, which greatly facilitates the
testing because
each clinical sample can be tested with universal, species-specific, genus-
specific,
antibiotic resistance gene and toxin gene PCR primers under uniform cycling
conditions. Furthermore, various combinations of primer pairs may be used in
multiplex PCR assays.
It is also an object of the present invention that tuf sequences, atpD
sequences
and recA sequences could serve as drug targets and these sequences and means
to
obtain them revealed in the present invention can assist the screening, design
and
modeling of these drugs.
CA 02307010 2000-OS-19
It is also an object of the present invention that tuf sequences, atpD
sequences
and recA sequences could serve for vaccine purposes and these sequences and
means
to obtain them revealed in the present invention can assist the screening,
design and
modeling of these vaccines.
We aim at developing a rapid test or kit (universal) to discard rapidly all
the
samples which are negative for bacterial, fungal or parasitical cells. This
test could be
used alone or combined with more specific identification to detect and
identify the
above bacterial and/or fungal and/or parasitical species and genera and to
determine
to rapidly the bacterial resistance to antibiotics and/or presence of
bacterial toxins.
Although the sequences from the selected antibiotic resistance genes are
available
from public databases and have been used to develop DNA-based tests for their
detection, our approach is unique because it represents a major improvement
over
current diagnostic methods based on bacterial cultures. Using an amplification
method for the simultaneous bacterial detection and identification and
antibiotic
resistance genes detection, there is no need for culturing the clinical sample
prior to
testing. Moreover, a modified PCR protocol has been developed to detect all
target
DNA sequences in approximately one hour under uniform amplification
conditions.
This procedure will save lives by optimizing treatment, will diminish
antibiotic
resistance because less antibiotics will be prescribed, will reduce the use of
broad
spectrum antibiotics which are expensive, decrease overall health care costs
by
preventing or shortening hospitalizations, and decrease the time and costs
associated
with clinical laboratory testing.
The diagnostic kits, primers and probes mentioned above can be used to
identify bacteria, fungi, parasites, antibiotic resistance genes and toxin
genes on any
type of sample, whether said diagnostic kits, primers and probes are used for
in vitro
or in situ applications. The said samples may include but are not limited to:
any
clinical sample, any environment sample, any microbial culture, any microbial
colony, any tissue, and any cell line.
It is also an object of the present invention that said diagnostic kits,
primers
and probes can be used alone or in conjunction with any other assay suitable
to
identify microorganisms, including but not limited to: any immunoassay, any
enzymatic assay, any biochemical assay, any lysotypic assay, any serological
assay,
any differential culture medium, any enrichment culture medium, any selective
culture medium, any specific assay medium, any identification culture medium,
any
enumeration cuture medium, any cellular stain, any culture on specific cell
lines, and
any infectivity assay on animals.
In the methods and kits described herein below, the oligonucleotide probes and
amplification primers have been derived from larger sequences (i.e. DNA
fragments
of at least 100 base pairs). All DNA fragments have been obtained either from
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CA 02307010 2000-OS-19
proprietary fragments or from public databases. DNA fragments selected from
public
databases are newly used in a method of detection according to the present
invention,
since they have been selected for their diagnostic potential.
In another embodiment, the amino acid sequences translated from the repertory
of tuf, atpD and recA sequences are also an object of the present invention.
It is clear to the individual skilled in the art that other oligonucleotide
sequences appropriate for (i) the universal bacterial detection, (ii) the
detection and
identification of the above microbial species or genus or family or group, and
(iii) the
detection of antibiotic resistance genes, and (iv) the detection of toxin
genes other
than those listed in Annexes I to III and XXI may also be derived from the
proprietary fragments or selected public databases sequences. For example, the
oligonucleotide primers or probes may be shorter or longer than the ones we
have
chosen; they may also be selected anywhere else in the proprietary DNA
fragments or
in the sequences selected from public databases; they may be also variants of
the
same oligonucleotide. If the target DNA or a variant thereof hybridizes to a
given
oligonucleotide, or if the target DNA or a variant thereof can be amplified by
a given
oligonucleotide PCR primer pair, the converse is also true; a given target DNA
may
hybridize to a variant oligonucleotide probe or be amplified by a variant
oligonucleotide PCR primer. Alternatively, the oligonucleotides may be
designed
from any DNA fragment sequences for use in amplification methods other than
PCR.
Consequently, the core of this invention is the identification of universal,
species-
specific, genus-specific, resistance gene-specific, toxin gene-specific
genomic or non-
genomic DNA fragments which are used as a source of specific and ubiquitous
oligonucleotide probes andlor amplification primers. Although the selection
and
evaluation of oligonucleotides suitable for diagnostic purposes requires much
effort,
it is quite possible for the individual skilled in the art to derive, from the
selected
DNA fragments, oligonucleotides other than the ones listed in Annexes I to III
and
XXI which are suitable for diagnostic purposes. When a proprietary fragment or
a
public databases sequence is selected for its specificity and ubiquity, it
increases the
3o probability that subsets thereof will also be specific and ubiquitous.
Since a high percentage of clinical specimens are negative for bacteria (Table
3), DNA fragments having a high potential for the selection of universal
oligonucleotide probes or primers were selected from proprietary and public
databases sequences. The amplification primers were selected from genes highly
conserved in bacteria, fungi and parasites, and are used to detect the
presence of any
bacterial or fungal or parasitical pathogen in clinical specimens in order to
determine
rapidly (less than one hour) whether it is positive or negative for bacteria,
fungi or
parasites. The selected genes, designated tuf, atpD and recA, encode
respectively 2
proteins (elongation factors Tu and G) involved in the translational process
during
4o protein synthesis, a protein (beta subunit) responsible for the catalytic
activity of
12
CA 02307010 2000-OS-19
proton pump ATPase and a protein responsible for the homologous recombination
of
genetic material. The alignments of tuf, atpD and recA sequences used to
derive the
universal primers include both proprietary and public databases sequences. The
universal primer strategy allows the rapid screening of the numerous negative
clinical
specimens (around 80% of the specimens received, see Table 3) submitted for
bacteriological testing.
Table 4 provides a list of the bacterial, fungal and parasitical species for
which
tuf and/or atpD and/or recA sequences are revealed in the present invention.
Tables 5
and 6 provide a list of antibiotics resistance genes and toxin genes selected
for
diagnostic purposes. Table 7 provides the origin of tuf, atpD and recA
sequences
listed in the sequence listing. Tables 8-10 provide lists or species used to
test
specificity and ubiquity of some assays described in examples.
13
CA 02307010 2000-OS-19
DETAILED DESCRIPTION OF THE INVENTION
HIGHLY CONSERVED GENES AND THEIR USE TO GENERATE SPECIES-
SPECIFIC, GENUS-SPECIFIC, FAMILY-SPECIFIC, GROUP-SPECIFIC AND
UNIVERSAL NUCLEIC ACID PROBES AND AMPLIFICATION PRIMERS TO
RAPIDLY DETECT AND IDENTIFY BACTERIAL, FUNGAL AND PARASITICAL
PATHOGENS FROM CLINICAL SPECIMENS FOR DIAGNOSIS
The present inventors compared the published Haemophilus influenzae and
Mycoplasma genitalium genomes and searched for the most conserved genes, which
would then serve, as paradigm to develop primers and probes. This sequence
comparison is highly informative as these two bacteria are distantly related
and most
genes present in the minimal genome of M. genitalium are likely to be present
in
every bacterium. Therefore genes conserved between these two bacteria are
likely to
be conserved in all other bacteria.
Following the genomic comparison, it was found that several protein-coding
genes were conserved in evolution. Highly conserved proteins included the
translation elongation factor Tu (EF-Tu) and the (3 subunit of FOF1 type ATP-
synthase, and to a lesser extent, the RecA recombinase.
Translation elongation factor Tu is a member of a family of GTP-binding
proteins which intervene in the interactions of tRNA molecules with the
ribosome
machinery during essential steps of protein synthesis. The role of elongation
factor
Tu is to facilitate the binding of aminoacylated tRNA molecules to the A site
of the
ribosome. The eukaryotic and archaebacterial homolog of EF-Tu is called
elongation
factor 1 alpha (EF-1a). All protein synthesis factors originated from a common
ancestor via gene duplications and fusions (Cousineau et al., 1997, J. Mol.
Evol.
45:661-670). In particular, elongation factor G (EF-G), although having a
functional
role in promoting the translocation of aminoacyl-tRNA molecules from the A
site to
the P site of the ribosome, shares sequence homologies with EF-Tu and is
thought to
have arisen from the duplication and fusion of an ancestor of the EF-Tu gene.
In addition, EF-Tu is known to be the target for antibiotics belonging to the
elfamycin's group as well as to other structural classes (Anborgh and
Parmeggiani,
1991, EMBO J. 10:779-784; Luiten et al., 1992, European patent application
serial
No. EP 0 466 251 A1). EF-G for its part, is the target of the antibiotic
fusidic acid. In
addition to its crucial activities in translation, EF-Tu has chaperone-like
functions in
protein folding, protection against heat denaturation of proteins and
interactions with
unfolded proteins (Caldas et al., 1998, J. Biol. Chem 273:11478-11482).
Interestingly, a form of the EF-Tu protein has been identified as a dominant
4o component of the periplasm of Neisseria gonorrhoeae (Porcella et al., 1996,
Microbiology 142:2481-2489), hence suggesting that at least in some bacterial
species, EF-Tu might be an antigen with vaccine potential.
FOF1 type ATP-synthase belongs to a superfamily of proton-translocating
ATPases divided in three major families: P, V and F (Nelson and Taiz,
14
CA 02307010 2000-OS-19
1989, TIBS 14:113-116). P-ATPases (or E1-E2 type) operate via a phosphorylated
intermediate and are not evolutionarily related to the other two families. V-
ATPases
(or VOV 1 type) are present on the vacuolar and other endomembranes of
eukaryotes,
on the plasma membrane of archaebacteria and also on the plasma membrane of
some
eubacteria especially species belonging to the order Spirochaetales as well as
to the
Chlamydiaceae and Deinococcaceae families. F-ATPases (or FOF1 type) are found
on the plasma membrane of most eubacteria, on the inner membrane of
mitochondria
and on the thylakoid membrane of chloroplasts. They function mainly in ATP
synthesis. They are large multimeric enzymes sharing numerous structural and
functional features with the V-ATPases. F and V-type ATPases have diverged
from a
common ancestor in an event preceding the appearance of eukaryotes. The (3
subunit
of the F-ATPases is the catalytic subunit and it possesses low but significant
sequence homologies with the catalytic A subunit of V-ATPases.
The translation elongation factors EF-Tu, EF-G and EF-loc, and the catalytic
subunit of F or V-types ATP-synthase, are highly conserved proteins sometimes
used
for phylogenetic analysis and their genes are also known to be highly
conserved
(Iwabe et al., 1989, Proc. Natl. Acad. Sci. USA 86:9355-9359, Gogarten et al.,
1989,
Proc. Natl. Acad. Sci. USA 86:6661-6665, Ludwig et al., 1993, Antonie van
Leeuwenhoek 64:285-305). A recent BLAST (Altschul et al., 1997, J. Mol. Biol.
215:403-410) search performed by the present inventors on the GenBank, EMBL,
DDBJ and specific genome project databases indicated that throughout bacteria,
the
EF-Tu and the (3 subunit of FOF1 type ATP-synthase genes may be more conserved
than other genes that are well conserved between H. influenzae and M.
genitalium.
The RecA recombinase is a multifunctional protein encoded by the recA gene.
It plays a central role in homologous recombination, it is critical for the
repair of
DNA damage and it is involved in the regulation of the SOS system by promoting
the
proteolytic digestion the LexA repressor. It is highly conserved in bacteria
and could
serve as a useful genetic marker to reconstruct bacterial phylogeny (Miller
and
Kokjohn, 1990, Annu. Rev. Microbiol. 44:365-394). Althought recA possesses
some
highly conserved sequence segments that we used to design universal primers
aimed
at sequencing the recA fragments, it is clearly not as well conserved as tuf
and atpD.
Hence, recA may not be optimal for universal detection of bacteria with high
sensitivity but it was chosen beacuse preliminary data indicated that tuf and
atpD may
sometimes be too closely related to find specific primer pairs that could
discriminate
between certain very closely related species and genera. While RecA, like tuf
and
atpD, possesses highly conserved regions suitable for the design of universal
sequencing primers, the less conserved region between primers should be
divergent
enough to allow species-specific and genus-specific primers in those cases.
Thus, as targets to design primers and probes for the genetic detection of
microorganisms, the present inventors have focused on the genes encoding these
three proteins: tuf, the gene for elongation factor Tu; atpD, the gene for (3
subunit of
FOF1 type ATP-synthase; and recA, the gene encoding the RecA recombinase. In
several bacterial genomes tuf is often found in two highly similar duplicated
copies
named tufA and tufB (Filer and Furano, 1981, J. Bacteriol. 148:1006-1011, Sela
et al.,
CA 02307010 2000-OS-19
1989, J. Bacteriol. 171:581-584). In some particular cases, more divergent
copies of
the tuf genes can exist in some bacterial species such as some Actinomycetes
(Luiten
et al. European patent application publication No. EP 0 446 251 A 1;
Vijgenboom et
al., 1994, Microbiology 140:983-998) and, as revealed as part of this
invention, in
several enterococcal species. In several bacterial species, tuf is organized
in an
operon with its homolog gene for the elongation factor G (EF-G) encoded by the
fusA gene. This operon is often named the str operon. The tuf, atpD and recA
genes
were chosen as they are well conserved in evolution and have highly conserved
stretches as well as more variable segments. Moreover, these three genes have
to eukaryotic orthologs which are described in the present invention as
targets to
identify fungi and parasites. The eukaryotic homolog of elongation factor Tu
is called
elongation factor 1-alpha (EF-1 oc) (gene name: tef, tefl , efl , ef I or EF-I
). In fungi,
the gene for EF-1 oc occurs sometimes in two or more highly similar duplicated
copies
(often named tefl, tef2, tef3...). In addition, eukaryotes have a copy of
elongation
factor Tu which is originating from their organelle genome ancestry (gene
name: tufl,
tufts or tufA). For the purpose of the current invention, the genes for these
four
functionally and evolutionarily linked elongation factors (bacterial EF-Tu and
EF-G,
eukaryotic EF-la, and organellar EF-Tu) will hereafter be designated as «tuf
sequences. The eukaryotic (mitochondrial) FOF1 type ATP-synthase beta subunit
gene is named atp2 in yeast. For the purpose of the current invention, the
genes of
catalytic subunit of either F or V-type ATP-synthase will hereafter be
designated as
«atpD sequences». The eukaryotic homologs of RecA are distributed in two
families,
typified by the RadS 1 and Dmc 1 proteins. For the purpose of the current
invention,
the genes corresponding to the latter proteins will hereafter be designated as
«recA
sequences.
Analysis of multiple sequence alignments of tuf and atpD sequences permitted
the design of oligonucleotide primers (and probes) capable of amplifying (or
hybridizing to) segments of tuf and atpD genes from a wide variety of
bacterial
species (see Examples 1 to 4 and Table 7). Sequencing primer pairs for tuf
sequences
are listed in Annex I and hybridization probes are listed in Annex III.
Sequencing
primer pairs for atpD sequences are listed in Annex II. Analysis of the main
subdivisions of tuf and atpD sequences (see Figures 1 and 2) permitted to
design
sequencing primers amplifying specifically each of these subdivisions. It
should be
noted that these sequencing primers could also be used as universal primers.
However, since some of these sequencing primers include several variable
sequence
(degenerated) positions, their sensitivity could be lower than that of
universal primers
developed for diagnostic purposes. Further subdivisions could be done on the
basis of
the various phyla where these genes are encountered.
Similarly, analysis of multiple sequence alignments of recA sequences present
in
the public databases permitted the design of oligonucleotide primers capable
of
amplifying segments of recA genes from a wide variety of bacterial species.
Sequencing primer pairs for recA sequences are listed in Annex XXI. The main
subdivisions of recA sequences comprise recA, rad51 and dmcl. Further
subdivisions
could be done on the basis of the various phyla where these genes are
encountered.
16
CA 02307010 2000-OS-19
The present inventor's strategy is to get as much sequence data information
from
the three conserved genes (tuf, atpD and recA). This ensemble of sequence data
forming a repertory (with subrepertories corresponding to each target gene and
their
main sequence subdivisions) and then using the sequence information of the
sequence
repertory (or subrepertories) to design primer pairs that could permit either
universal
detection of bacteria or fungi or parasites, detection of a family or group of
microorganism (e.g. Enterobacteriaceae), detection of a genus (e.g.
Streptococcus) or
finally a specific species (e.g. Staphylococcus aureus). It should be noted
that for the
purpose of the present invention a group of microorganisms is defined
depending on
the needs of the particular diagnostic test. It does not need to respect a
particular
taxonomical grouping or phylum. See example 12 where primers were designed to
amplify a group a bacteria consisting of the 17 major bacterial species
encountered as
contaminants of platelet concentrates. Also remark that in that example, the
primers'
specificity is not perfect since the objective of that particular test is to
be able to
sensitively and rapidly detect at least the 17 important bacterial species,
but the
primers could also detect other species as well, as shown in Table 14. In
these
circumstances the primers shown in example 12 are considered universal for
platelet-
contaminating bacteria. To develop an assay specific for the latter, one or
more
primers or probes specific to each species could be designed. Another example
of
primers and/or probes for group detection is given by the Pseudomonad group
primers. These primers were designed based upon alignment of tuf sequences
from
real Pseudomonas species as well as from former Pseudomonas species such as
Stenotrophomonas maltophilia. The resulting primers are able to amplify all
Pseudomonas species tested as well as several species belonging to different
genera,
hence as being specific for a group including Pseudomonas and other species,
we
defined that group as Pseudomonads as several members were former Pseudomonas.
For certain applications, it may be possible to develop a universal, group,
family
or genus-specific reaction and to proceed to species identification using
sequence
information within the amplicon to design species-specific internal probes or
primers,
or alternatively, to proceed directly by sequencing the amplicon. The various
strategies will be discussed further below.
The ensembles formed by public and proprietary tuf, atpD and recA sequences
are used in a novel fashion so they constitute three databases containing
useful
information for the identification of microorganisms.
Oligonucleotide primers and probes design and synthesis
The tuf, atpD and recA DNA fragments sequenced by us or selected from public
databases (GenBank and EMBL) were used to design oligonucleotides primers and
probes for diagnostic purposes. We also relied on the corresponding peptide
sequence
of tuf, atpD and recA sequences to facilitate the identification of regions
suitable for
primers and probes design. As part of the design rules, all oligonucleotides
(probes
for hybridization and primers for DNA amplification) were evaluated for their
17
CA 02307010 2000-OS-19
suitability for hybridization or DNA amplification by polymerise chain
reaction
(PCR) by computer analysis using standard programs (i.e. the Genetics Computer
Group (GCG) programs and the primer analysis software OligoTM 5.0). The
potential
suitability of the PCR primer pairs was also evaluated prior to the synthesis
by
verifying the absence of unwanted features such as long stretches of one
nucleotide
and a high proportion of G or C residues at the 3' end (Persing et al., 1993,
Diagnostic Molecular Microbiology: Principles and Applications, American
Society
for Microbiology, Washington, D.C.). Oligonucleotide probes and amplification
primers were synthesized using an automated DNA synthesizer (Perkin-Elmer
Corp.,
Applied Biosystems Division).
The oligonucleotide primers or probes may be derived from either strand of the
duplex DNA. The primers or probes may consist of the bases A, G, C, or T or
analogs
and they may be degenerated at one or more chosen nucleotide position(s). The
primers or probes may be of any suitable length and may be selected anywhere
within
the DNA sequences from proprietary fragments or from selected database
sequences
which are suitable for (i) the universal detection of bacteria or fungi or
parasites, (ii)
the species-specific detection and identification of any microorganism,
including but
not limited to: Candida albicans, Candida dubliniensis, Chlamydia pneumoniae,
Chlamydia trachomatis, Cryptosporidium parvum, Enterococcus faecalis,
Enterococcus faecium, Enterococcus gallinarum, Escherichia coli, Haemophilus
influenzae, Neisseria gonorrhoeae, Staphylococcus aureus, Staphylococcus
epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis,
Staphylococcus
saprophyticus, Streptococcus agalactiae, Trypanosoma brucei, Trypanosoma
cruzi,
(iii) the genus-specific detection of Bordetella species, Candida species,
Clostridium
species, Corynebacterium species, Entamoeba species, Enterococcus species,
Giardia species, Leishmania species, Staphylococcus species, Streptococcus
species,
Trypanosoma species, (iv) the family-specific detection of Enterobacteriaceae
family
members, Mycobacteriaceae family members, (v) the detection of Enterococcus
casseliflavus-flavescens-gallinarum group, Trypanosomatidae family,
Pseudomonads
extended group, Platelet-contaminating bacteria group, or (vi) the detection
of
clinically important antibiotic resistance genes listed in Table 5, or (vii)
the detection
of clinically important toxin genes listed in Table 6.
Variants for a given target bacterial gene are naturally occurring and are
attributable to sequence variation within that gene during evolution (Watson
et al.,
1987, Molecular Biology of the Gene, 4'h ed., The Benjamin/Cummings Publishing
Company, Menlo Park, CA; Lewin, 1989, Genes IV, John Wiley & Sons, New York,
NY). For example, different strains of the same bacterial species may have a
single or
more nucleotide variations) at the oligonucleotide hybridization site. The
person
skilled in the art is well aware of the existence of variant bacterial or
fungal DNA
sequences for a specific gene and that the frequency of sequence variations
depends
18
CA 02307010 2000-OS-19
on the selective pressure during evolution on a given gene product. The
detection of a
variant sequence for a region between two PCR primers may be demonstrated by
sequencing the amplification product. In order to show the presence of
sequence
variants at the primer hybridization site, one has to amplify a larger DNA
target with
PCR primers outside that hybridization site. Sequencing of this larger
fragment will
allow the detection of sequence variation at this site. A similar strategy may
be
applied to show variants at the hybridization site of a probe. Insofar as the
divergence
of the target sequences or a part thereof does not affect the specificity and
ubiquity of
the amplification primers or probes, variant bacterial DNA is under the scope
of this
invention. Variants of the selected primers or probes may also be used to
amplify or
hybridize to a variant DNA.
Sepuencin~ of tuf seguences from a variety of bacterial, fungal and
narasitical
s ecies
The nucleotide sequence of a portion of tuf sequences was determined for a
variety of bacterial, fungal and parasitical species. The amplification
primers (SEQ
ID NOs. 107 and 108 in previous patent application W098/20157), which amplify
a
tuf gene portion of approximately 890 bp, were used along with newly designed
sequencing primer pairs (See Annex I for the sequencing primers for tuf
sequences).
Most primer pairs can amplify different copies of tuf genes (tufA and tufB).
This is
not surprising since it is known that for several bacterial species these two
genes are
nearly identical. For example, the entire tufA and tufB genes from E, coli
differ at
only 13 nucleotide positions (Neidhardt et al., 1996, Escherichia coli and
Salmonella:
Cellular and Molecular Biology, 2°d ed., American Society for
Microbiology Press,
Washington, D.C.). Similarly, some fungi are known to have two nearly
identical
copies of tuf sequences (EF-la). These amplification primers are degenerated
at
several nucleotide positions and contain inosines in order to allow the
amplification
of a wide range of tuf sequences. The strategy used to select these
amplification
primers is similar to that illustrated in Annex I for the selection of
universal primers.
The tuf sequencing primers even sometimes amplified highly divergent copies of
tuf
genes (tuff) as illustrated in the case of some enterococcal species (SEQ ID
NOs.:
73, 75, 76, 614 to 618, 621 and 987 to 989). To prove this we first had to
clone PCR
products before being able to sequence them. Using the cloned sequence data we
designed a new pair of sequencing primers specific to the divergent (tuff)
copy of
enterococci (SEQ ID NOs.: 658-659 and 661) and then sequenced directly the
tuff
amplicons. The amplification primers (SEQ ID NOs.: 543, 556, 557, 660, 664,
694,
696 and 697) could be used to amplify the tuf sequences from any bacterial
species.
The amplification primers (SEQ ID NOs.: 558, 559, 560, 653, 654, 655, 813 and
815)
could be used to amplify the tuf (EF-1 oc) genes from any fungal and
parasitical
19
CA 02307010 2000-OS-19
species. The amplification primers SEQ ID NOs. 1221-1228 could be used to
amplify
bacterial tuf sequences of the EF-G subdivision (fusA) (Figure 3). The
amplification
primer SEQ ID NOs 1224, and 1227-1229 could be used to amplify bacterial tuf
sequences comprising the end of EF-G (ficsA) and the beginning of EF-Tu (tub,
as
shown in Figure 3.
The tuf fragments to be sequenced were amplified using the following
amplification protocol: One ~,l of cell suspension (or of purified genomic DNA
0.1-
0.5 ng/~,1) was transferred directly to 19 ~,l of a PCR reaction mixture. Each
PCR
reaction contained 50 mM KCI, 10 mM Tris-HCl (pH 9.0), 0.1 % Triton X-100, 2.5
l0 mM MgCl2, 1 ~,M of each of the 2 primers, 200 ~M of each of the four dNTPs,
0.5
unit of Taq DNA polymerase (Promega Corp., Madison, WI). PCR reactions were
subjected to cycling using a PTC-200 thermal cycler (MJ Research Inc.,
Watertown,
Mass.) as follows: 3 min at 96 °C followed by 30-45 cycles of 1 min at
95 °C for the
denaturation step, 1 min at 50-55 °C for the annealing step and 1 min
at 72 °C for the
extension step. Subsequently, twenty microliters of the PCR-amplified mixture
were
resolved by electrophoresis in a 1.5% agarose gel. The amplificon was then
visualized by staining with methylene blue (Flores et al., 1992,
Biotechniques,
13:203-205). The size of the amplification products was estimated by
comparison
with a 100-by molecular weight ladder. The band corresponding to the specific
amplification product was excised from the agarose gel and purified using the
QIAquickTM gel extraction kit (QIAGEN Inc., Chatsworth, CA). The gel-purified
DNA fragment was then used directly in the sequencing protocol. Both strands
of the
tuf genes amplification product were sequenced by the dideoxynucleotide chain
termination sequencing method by using an Applied Biosystems automated DNA
sequencer (model 377) with their Big DyeTM Terminator Cycle Sequencing Ready
Reaction Kit (Perkin-Elmer Corp., Applied Biosystems Division, Foster City,
CA).
The sequencing reactions were performed by using the same amplification
primers
and 10 ng/100 by of the gel-purified amplicon per reaction. For the sequencing
of
long amplicons such as those of eukaryotic tuf (EF-1 oc) sequences, we
designed
internal sequencing primers (SEQ ID NOs.: 654, 655 and 813) to be able to
obtain
sequence data on both strands for most of the fragment length. In order to
ensure that
the determined sequence did not contain errors attributable to the sequencing
of PCR
artefacts, we have sequenced two preparations of the gel-purified tuf
amplification
product originating from two independent PCR amplifications. For most target
microbial species, the sequences determined for both amplicon preparations
were
identical. In case of discrepancies, a third independant PCR amplification was
sequenced. Furthermore, the sequences of both strands were 100% complementary
thereby confirming the high accuracy of the determined sequence. The tuf
sequences
determined using the above strategy are described in the Sequence Listing.
Table 7
gives the originating microbial species and the source for each tuf sequence
in the
CA 02307010 2000-OS-19
Sequence Listing.
The alignment of the tuf sequences determined by us or selected from
databases reveals clearly that the length of the sequenced portion of the tuf
genes is
variable. There may be insertions or deletions of several amino acids. In
addition, in
several fungi introns were observed. Intron sequences are part of tuf
sequences and
could be useful in the design of species-specific primers and probes. This
explains
why the size of the sequenced tuf amplification products was variable from one
species to another. Consequently, the nucleotide positions indicated on top of
each of
Annexes IV to XXI, XXIII to XXXI, XXXVIII and XLII do not correspond for
sequences having insertions or deletions.
It should also be noted that the various tuf sequences determined by us
occasionally contain degenerescences. These degenerated nucleotides correspond
to
sequence variations between tufA and tufB genes (or copies of the EF-G
subdivision
of tuf sequences, or copies of EF-1 cc subdivision of tuf sequences for fungi
and
parasites) because the amplification primers amplify both tuf genes. These
nucleotide
variations were not attributable to nucleotide misincorporations by the Taq
DNA
polymerise because the sequence of both strands was identical and also because
the
sequences determined with both preparations of the gel-purified tuf amplicons
were
identical.
The selection of amplification primers from tuf seguences
The tuf sequences determined by us or selected from public databases were used
to select PCR primers for (i) the universal detection of bacteria, (ii) the
genus-specific
detection and identification of Enterococcus sp. and Staphylococcus sp. and
(iii) the
species-specific detection and identification of Candida albicans. The
strategy used
to select these PCR primers was based on the analysis of multiple sequence
alignments of various tuf sequences. For more details about the selection of
PCR
primers from tuf sequences please refer to Examples and Annexes.
Secruencin~ of att~D and recA seguences from a variety of bacterial, fungal
and
parasitical species
The method use to obtain atpD and recA sequences is similar to that described
above for tuf sequences.
The selection of amplification primers from atnD or recA
The comparison of the nucleotide sequence for the atpD or recA genes from
21
CA 02307010 2000-OS-19
various bacterial fungal and parasitical species allowed the selection of PCR
primers
(refer to Examples 1, 2 and 6 and Annexes IV, V, X, XX, XXI).
DNA amplification
For DNA amplification by the widely used PCR (polymerase chain reaction)
method, primer pairs were derived from proprietary DNA fragments or from
database
sequences. Prior to synthesis, the potential primer pairs were analyzed by
using the
OligoTM 5.0 software to verify that they were good candidates for PCR
amplification.
During DNA amplification by PCR, two oligonucleotide primers binding
respectively to each strand of the heat-denatured target DNA from the
bacterial
genome are used to amplify exponentially in vitro the target DNA by successive
thermal cycles allowing denaturation of the DNA, annealing of the primers and
synthesis of new targets at each cycle (Persing et al, 1993, Diagnostic
Molecular
Microbiology: Principles and Applications, American Society for Microbiology,
Washington, D.C.).
Briefly, the PCR protocols were as follows: Treated clinical specimens or
standardized bacterial or fungal or parasitical suspensions (see below) or
purified
genomic DNA from bacteria, fungi or parasites were amplified in a 20 ~,1 PCR
reaction mixture. Each PCR reaction contained 50 mM KCI, 10 mM Tris-HCl (pH
9.0), 2.5 mM MgCl2, 0.4 ~.M of each primer, 200 ~.M of each of the four dNTPs
and
0.5 unit of Taq DNA polymerase (Promega) combined with the TaqStartTM antibody
(Clontech Laboratories Inc., Palo Alto, CA). The TaqStartTM antibody, which is
a
neutralizing monoclonal antibody to Taq DNA polymerase, was added to all PCR
reactions to enhance the specificity and the sensitivity of the amplifications
(Kellogg
et al., 1994, Biotechniques 16:1134-1137). The treatment of the clinical
specimens
varies with the type of specimen tested, since the composition and the
sensitivity
level required are different for each specimen type. It consists in a rapid
protocol to
lyse the bacterial cells and eliminate the PCR inhibitory effects. For
amplification
from bacterial or fungal cultures or from purified genomic DNA, the samples
were
added directly to the PCR amplification mixture without any pre-treatment
step. An
internal control was derived from sequences not found in the target
microorganisms
or in the human genome. The internal control was integrated into all
amplification
reactions to verify the efficiency of the PCR assays and to ensure that
significant
PCR inhibition was absent. Alternatively, an internal control derived from
rRNA was
also useful to monitor the efficiency of microbial lysis protocols.
PCR reactions were then subjected to thermal cycling (3 min at 95 °C
followed
by 30 cycles of 1 second at 95 °C for the denaturation step and 30
seconds at 50-65
°C for the annealing-extension step) using a PTC-200 thermal cycler (MJ
Research
Inc.). The number of cycles performed for the PCR assays varies according to
the
22
CA 02307010 2000-OS-19
sensitivity level required. For example, the sensitivity level required for
microbial
detection directly from clinical specimens is higher for blood specimens than
for
urine specimens because the concentration of microorganisms associated with a
septicemia can be much lower than that associated with a urinary tract
infection.
Consequently, more sensitive PCR assays having more thermal cycles are
required
for direct detection from blood specimens. Similarly, PCR assays performed
directly
from bacterial or fungal or parasitical cultures may be less sensitive than
PCR assays
performed directly from clinical specimens because the number of target
organisms is
normally much lower in clinical specimens than in microbial cultures.
The person skilled in the art of DNA amplification knows the existence of
other rapid amplification procedures such as ligase chain reaction (LCR),
transcription-mediated amplification (TMA), self-sustained sequence
replication
(3SR), nucleic acid sequence-based amplification (NASBA), strand displacement
amplification (SDA), branched DNA (bDNA), cycling probe technology (CPT),
solid
phase amplification (SPA), rolling circle amplification technology (RCAT), and
nuclease dependent signal amplification (NDSA) (Lee et al., 1997, Nucleic Acid
Amplification Technologies: Application to Disease Diagnosis, Eaton
Publishing,
Boston, MA; Persing et al., 1993, Diagnostic Molecular Microbiology:
Principles and
Applications, American Society for Microbiology, Washington, D.C.). The scope
of
this invention is not limited to the use of amplification by PCR, but rather
includes
the use of any rapid nucleic acid amplification method or any other procedure
which
may be used to increase rapidity and sensitivity of the nucleic acid-based
tests. The
scope of the present invention also covers the use of any nucleic acids
amplification
and detection technology including real-time or post-amplification detection
technologies, any amplification technology combined with detection, any
hybridization nucleic acid chips or arrays technologies, any amplification
chips or
combination of amplification and hybridization chips technologies. Detection
and
identification by any sequencing method is also under the scope of the present
invention.
Any oligonucleotide suitable for the amplification of nucleic acids by
approaches other than PCR or for DNA hybridization and derived from the
species-
specific, genus-specific and universal DNA fragments as well as from selected
antibiotic resistance or toxin gene sequences included in this document are
also under
the scope of this invention.
Detection of amulification products
Classically, detection of amplification is performed by standard ethidium
bromide-stained agarose gel electrophoresis. It is clear that other methods
for the
detection of specific amplification products, which may be faster and more
practical
for routine diagnosis, may be used. Such methods may be based on the detection
of
fluorescence after or during amplification. One simple method for monitoring
23
CA 02307010 2000-OS-19
amplified DNA is to measure its rate of formation by measuring the increase in
fluorescence of intercalating agents such as ethidium bromide or SYBR~ Green
I. If
more specific detection is required, fluorescence-based technologies can
monitor the
appearance of a specific product during the reaction. The use of dual-labeled
fluorogenic probes such as in the TaqManTM system which utilizes the 5'-3'
exonuclease activity of the Taq polymerase is a good example (Livak K.J. et
al. 1995,
PCR Methods Appl. 4:357-362). TaqManTM can be performed during amplification
and this "real-time" detection can be done in a single closed tube hence
eliminating
post-PCR sample handling and consequently preventing the risk of amplicon
carryover (TaqManTM system from Perkin Elmer or AmplisensorTM from
Biotronics).
Several other fluorescence-based detection methods can be performed in real-
time.
Fluorescence resonance energy transfer (FRET) is the principle behind the use
of
adjacent hybridization probes and molecular beacons. Adjacent hybridization
probes
are designed to be internal to the amplification primers. The 3' end of one
probe is
labelled with a donor fluorophore while the 5' end of an adjacent probe is
labelled
with an acceptor fluorophore. When the two probes are specifically hybridized
in
closed proximity (spaced by 1 to 5 nucleotides) the donor fluorophore which
has been
excited by an external light source emits light that is absorbed by a second
acceptor
that emit more fluorescence and yields a FRET signal. Molecular beacons
possess a
stem-and-loop structure where the loop is the probe and at the bottom of the
stem a
fluorescent moiety is at one end while a quenching moiety is at the other end.
The
beacons undergo a fluorogenic conformational change when they hybridize to
their
targets hence separating the fluorochrome from its quencher. The FRET
principle is
also used in an air thermal cycler with a built-in fluorometer (Wittwer, C.T.
et al.
1997. BioTechniques 22:130-138). The amplification and detection are extremely
rapid as reactions are performed in capillaries : it takes 18 min to complete
45 cycles.
Those techniques are suitable especially in the case where few pathogens are
searched for. Boehringer-Roche Inc. sells the LightCyclerTM, an apparatus
capable of
rapid cycle PCR combined with fluorescent SYBR° Green I or FRET
detection. We
recently demonstrated in our laboratory, real-time detection of 10 CFU in less
than 40
minutes using adjacent hybridization probes on the LightCyclerTM. Methods
based on
the detection of fluorescence are particularly promising for utilization in
routine
diagnosis as they are very rapid, quantitative and can be automated.
Microbial pathogens detection and identification may also be performed by
solid
support or liquid hybridization using species-specific internal DNA probes
hybridizing to an amplification product. Such probes may be generated from any
sequences from our repertory and designed to specifically hybridize to DNA
amplification products which are objects of the present invention.
Alternatively, the
internal probes for species or genus or family or group detection and
identification
may be derived from the amplicons produced by a universal, family, group or
genus
24
CA 02307010 2000-OS-19
amplification assay. The oligonucleotide probes may be labeled with biotin or
with
digoxigenin or with any other reporter molecule (for more details see below
the
section on hybrid capture). Hybrization on a solid support is amendable to
miniaturization.
At present the oligonucleotide nucleic acid microarray technology is
appealing.
Currently, available low to medium density arrays (Heller et al., An
integrated
microelectronics hybridization system for genomic research and diagnostic
applications. In: Harrison, D.J., and van den Berg, A., 1998, Micro total
analysis
systems '98, Kluwer Academic Publisher, Dordrecht.) could specifically capture
l0 fluorescent-labelled amplicons. Detection methods for hybridization are not
limited
to fluorescence; potentiometry, colorimetry and plasmon resonance are some
examples of alternative detection methods. In addition to detection by
hybridization,
nucleic acid microarrays could be used to perform rapid sequencing by
hybridization.
Mass spectrometry could also be applicable for rapid identification of the
amplicon or
even for sequencing of the amplification products (Chiu and Cantor, 1999,
Clinical
Chemistry 45:1578; Berkenkamp et al., 1998, Science 281:260).
We also keep in mind the major challenge of molecular diagnostics tools, i.e.
integration of the major steps including sample preparation, genetic
amplification,
detection, data analysis and presentation (Anderson et al., Advances in
integrated
genetic analysis. In: Harrison, D.J., and van den Berg, A., 1998, Micro total
analysis
systems '98, Kluwer Academic Publisher, Dordrecht.).
To ensure PCR efficiency, glycerol, dimethyl sulfoxide (DMSO) or other related
solvents can be used to increase the sensitivity of the PCR and to overcome
problems
associated with the amplification of a target DNA having a high GC content or
forming strong secondary structures (Dieffenbach and Dveksler, 1995, PCR
Primer:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, New
York).
The concentration ranges for glycerol and DMSO are 5-15% (v/v) and 3-10%
(v/v),
respectively. For the PCR reaction mixture, the concentration ranges for the
amplification primers and MgCl2 are 0.1-1.5 ~,M and 1.0-10.0 mM, respectively.
Modifications of the standard PCR protocol using external and nested primers
(i.e.
nested PCR) or using more than one primer pair (i.e. multiplex PCR) may also
be
used (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and
Applications, American Society for Microbiology, Washington, D.C.). For more
details about the PCR protocols and amplicon detection methods, see Examples.
Hybrid capture and chemiluminescence detection of amplification products
Hybridization and detection of amplicons by chemiluminescence were adapted
from Nikiforov et al. (1994, PCR Methods and Applications 3:285-291 and 1995,
Anal. Biochem. 227:201-209 ) and from the DIGTM system protocol of Boehringer
Mannheim. Briefly, 50 ~,1 of a 25 picomoles solution of capture probe diluted
in EDC
{ 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride} are
immobilized in
CA 02307010 2000-OS-19
each well of 96-wells plates (MicroliteTM 2, Dynex) by incubation overnight at
room
temperature. The next day, the plates are incubated with a solution of 1% BSA
diluted into TNTw (10 mM Tris-HCI, pH 7.5; 150 mM NaCI; 0.05% TweenTM 20)
for 1 hour at 37 °C. The plates are then washed on a Wellwash AscentTM
(Labsystems) with TNTw followed by Washing Buffer (100 mM malefic acid pH7.5;
150 mM NaCI; 0.3 % TweenTM 20).
The amplicons were labelled with DIG-11-dUTP during PCR using the PCR
DIG Labelling Mix from Boehringer Mannheim according to the manufacturer's
instructions. Hybridization of the amplicons to the capture probes is
performed in
triplicate at stringent temperature (generally, probes are designed to allow
hybrization
at 55 °C, the stringent temperature) for 30 minutes in 1.5 M NaCI; 10
mM EDTA. It
is followed by two washes in 2 X SSC; 0.1% SDS, then by four washes in O.1X
SSC;
0.1% SDS at the stringent temperature (55 °C). Detection with 1,2
dioxetane
chemiluminescent alkaline phosphatase substrates like CSPD~ (Tropix inc.) is
performed according to the manufacturer's instructions but with shorter
incubations
times and a different antibody concentration. The plates are agitated at each
step, the
blocking incubation is performed for only 5 minutes, the anti-DIG-AP1 is used
at a
1:1000 dilution, the incubation with antibody lasts 15 minutes, the plates are
washed
twice for only 5 minutes. Finally, after a 2 minutes incubation into the
detection
buffer, the plates are incubated 5 minutes with CSPD~ at room temperature
followed
by a 10 minutes incubation at 37 °C without agitation. Luminous signal
detection is
performed on a Dynex Microtiter Plate Luminometer using RLU (Relative Light
Units).
Specificity and ubiquity tests for oligonucleotide primers and probes
The specificity of oligonucleotide primers and probes was tested by
amplification of DNA or by hybridization with bacterial or fungal or
parasitical
species selected from a panel comprising closely related species and species
sharing
the same anatomo-pathological site (see Annexes and Examples). All of the
bacterial,
fungal and parasitical species tested were likely to be pathogens associated
with
infections or potential contaminants which can be isolated from clinical
specimens.
Each target DNA could be released from bacterial cells using standard chemical
and/or physical treatments to lyse the cells (Sambrook et al., 1989, Molecular
Cloning: A Laboratory Manual, 2°d ed., Cold Spring Harbor Laboratory
Press, Cold
Spring Harbor, NY) or alternatively, genomic DNA purified with the GNOMETM
DNA kit (Bio101, Vista, CA) was used. Subsequently, the DNA was subjected to
amplification with the primer pairs.
Oligonucleotides primers found to amplify specifically the target species,
genus,
family or group were subsequently tested for their ubiquity by amplification
(i.e.
26
CA 02307010 2000-OS-19
ubiquitous primers amplified most or all isolates of the target species or
genus or
family or group). The specificity and ubiquity of the PCR assays using the
selected
amplification primer pairs were tested either directly from cultures of
microbial
species or from purified microbial genomic DNA.
Probes were tested in hybrid capture assays as described above. An
oligonucleotide probe was considered specific only when it hybridized solely
to DNA
from the species or genus or family or group from which it was selected.
Oligonucleotide probes found to be specific were subsequently tested for their
ubiquity (i.e. ubiquitous probes recognized most or all isolates of the target
species or
genus or family or group) by hybridization to microbial DNAs from different
clinical
isolates of the species or genus or family or group of interest including ATCC
reference strains. Similarly, oligonucleotide primers and probes could be
derived
from antibiotic resistance or toxin genes which are objects of the present
invention.
Reference strains
The reference strains used to built proprietary tuf, atpD and recA sequence
data
subrepertories, as well as to test the amplification and hybridization assays
were
obtained from (i) the American Type Culture Collection (ATCC), (ii) the
Laboratoire
de same publique du Quebec (LSPQ), (iii) the Centers for Disease Control and
Prevention (CDC), (iv) the National Culture Type Collection (NCTC) and (v)
several
other reference laboratories throughout the world. The identity of our
reference
strains was confirmed by phenotypic testing and reconfirmed by analysis of
tuf, atpD
and recA sequences (see example 13).
Antibiotic resistance genes
Antimicrobial resistance complicates treatment and often leads to therapeutic
failures. Furthermore, overuse of antibiotics inevitably leads to the
emergence of
bacterial resistance. Our goal is to provide clinicians, in approximately one
hour, the
needed information to prescribe optimal treatments. Besides the rapid
identification
of negative clinical specimens with DNA-based tests for universal bacterial
detection
and the identification of the presence of a specific pathogen in the positive
specimens
with species- and/or genus- and/or family- and/or group-specific DNA-based
tests,
clinicians also need timely information about the ability of the bacterial
pathogen to
resist antibiotic treatments. We feel that the most efficient strategy to
evaluate rapidly
bacterial resistance to antimicrobials is to detect directly from the clinical
specimens
the most common and clinically important antibiotic resistance genes (i.e. DNA-
based tests for the detection of antibiotic resistance genes). Since the
sequence from
the most important and common bacterial antibiotic resistance genes are
available
27
CA 02307010 2000-OS-19
from public databases, our strategy is to use the sequence from a portion or
from the
entire resistance gene to design specific oligonucleotide primers or probes
which will
be used as a basis for the development of rapid DNA-based tests. The list of
each of
the bacterial antibiotic resistance genes selected on the basis of their
clinical
relevance (i.e. high incidence and importance) is given in Table 5. Our
approach is
unique because the antibiotic resistance genes detection and the bacterial
detection
and identification can be performed simultaneously in multiplex assays under
uniform PCR amplification conditions. These amplifications can also be done
separately.
Toxin genes
Toxin identification is often very important to prescribe optimal treatments.
Besides the rapid identification of negative clinical specimens with DNA-based
tests
for universal bacterial detection and the identification of the presence of a
specific
pathogen in the positive specimens with species- and/or genus- and/or family-
and/or
group-specific DNA-based tests, clinicians sometimes need timely information
about
the ability of certain bacterial pathogens to produce toxins. Since the
sequence from
the most important and common bacterial toxin genes are available from public
databases, our strategy is to use the sequence from a portion or from the
entire toxin
gene to design specific oligonucleotide primers or probes which will be used
as a
basis for the development of rapid DNA-based tests. The list of each of the
bacterial
toxin genes selected on the basis of their clinical relevance (i.e. high
incidence and
importance) is given in Table 6. Our approach is unique because the toxin
genes
detection and the bacterial detection and identification can be performed
simultaneously in multiplex assays under uniform PCR amplification conditions.
Universal bacterial detection
In the routine microbiology laboratory, a high percentage of clinical
specimens
sent for bacterial identification are negative by culture. Testing clinical
samples with
universal amplification primers or universal probes to detect the presence of
bacteria
prior to specific identification and screening out the numerous negative
specimens is
thus useful as it reduces costs and may rapidly orient the clinical management
of the
patients. Several amplification primers and probes were therefore synthesized
from
highly conserved portions of bacterial sequences from the tuf and atpD
sequences.
The universal primers selection was based on a multiple sequence alignment
constructed with sequences from our repertory.
All computer analysis of amino acid and nucleotide sequences were performed
4o by using the GCG programs. Subsequently, optimal PCR primers for the
universal
28
CA 02307010 2000-OS-19
amplification of bacteria were selected with the help of the OligoTM program.
The
selected primers are degenerated at several nucleotide positions and contain
several
inosines in order to allow the amplification of all clinically relevant
bacterial species
(Annex I). Inosine is a nucleotide analog able to specifically bind to any of
the four
nucleotides A, C, G or T. Degenerated oligonucleotides consist of an
oligonucleotide
mix having two or more of the four nucleotides A, C, G or T at the site of
mismatches. The inclusion of inosine and/or of degenerescences in the
amplification
primers allow mismatch tolerance thereby permitting the amplification of a
wider
array of target nucleotide sequences (Dieffenbach and Dveksler, 1995 PCR
Primer: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, NY).
The amplification conditions with the universal primers are very similar to
those
used for the species- and genus-specific amplification assays except that the
annealing temperature is slightly lower. The original universal PCR assay
described
in our co-pending W098/20157 (SEQ ID NOs. 23-24 of the latter application) was
specific and nearly ubiquitous for the detection of bacteria. The specificity
for
bacteria was verified by amplifying genomic DNA isolated from the 12 fungal
species as well as genomic DNA from Leishmania donovani, Saccharomyces
cerevisiae and human lymphocytes. None of the above eukaryotic DNA
preparations
could be amplified by the universal assay, thereby suggesting that this test
is specific
for bacteria. The ubiquity of the universal assay was verified by amplifying
genomic
DNAs from 116 reference strains which represent 95 of the most clinically
relevant
bacterial species. These species have been selected from the bacterial species
listed in
Table 4. We found that at least 104 of these species could be amplified.
However, the
assay could be improved since bacterial species which could not be amplified
with
the original tuf sequences-based assay included species belonging to the
following
genera: Corynebacterium (11 species) and Stenotrophomonas (1 species).
Sequencing
of the tuf genes from these bacterial species and others has been performed in
the
scope of the present invention in order to improve the universal assay. This
sequencing data has been used to select new universal primers which may be
more
ubiquitous. Also, we improved our primer and probes design strategy by taking
into
consideration the phylogeny observed in analysing our repertory of tuf, atpD
and
recA sequences. Data from each of the 3 main subrepertories (tuf, atpD and
recA)
was subjected to a basic phylogenic analysis using the Pileup command from
version
10 of the GCG package (Genetics Computer Group, inc.). This analysis indicated
the
main branches or phyla reflecting the relationships between sequences. Instead
of
trying to design primers or probes able to hybridize to all phyla, we designed
primers
or probes able to hybridize to the main phyla while trying to use the largest
phylum
possible. This strategy should allow less degenerated primers hence improving
sensitivity and by combining primers in a mutiplex assay, improve ubiquity.
Universal primers SEQ ID NOs. 643-645 based on tuf sequences have been
designed
29
CA 02307010 2000-OS-19
to amplify most pathogenic bacteria except Actinomyceteae, Clostridiaceae and
the
Cytophaga, Flexibacter and Bacteroides phylum (pathogenic bacteria of this
phylum
include mostly Bacteroides, Porphyromonas and Prevotella species). Primers to
fill
these gaps have been designed for Actinomyceteae (SEQ ID NOs. 646-648),
Clostridiaceae (SEQ ID NOs. 796-797, 808-811 ), and the Cytophaga, Flexibacter
and Bacteroides phylum (SEQ ID NOs. 649-651). These primers sets could be used
alone or in conjuction to render the universal assay more ubiquitous. These
primers
are in the process of being tested.
Universal primers derived from atpD sequences include SEQ ID NOs. 562-565.
Combination of these primers does not amplify human DNA but should amplify
almost all pathogenic bacterial species except proteobacteria belonging to the
epsilon
subdivision (Campylobacter and Helicobacter), the bacteria from the Cytophaga,
Flexibacter and Bacteroides group and some actinomycetes and corynebacteria.
By
analysing atpD sequences from the latter species, primers and probes to
specifically
fill these gaps could be designed and used in conjuction with primers SEQ ID
NOs.
562-565. These primers are in the process of being tested.
In addition, universality of the assay could be expanded by mixing atpD
sequences-derived primers with tuf sequences-derived primers. Ultimately, even
recA
sequences-derived primers could be added to fill some gaps in the universal
assay.
It is important to note that the 95 bacterial species selected to test the
ubiquity of
the universal assay include all of the most clinically relevant bacterial
species
associated with a variety of human infections acquired in the community or in
hospitals (nosocomial infections). The most clinically important bacterial and
fungal
pathogens are listed in Tables 1 and 2.
Amino acid seguences derived from tuf, atpD and recA seguences
The amino acid sequences translated from the repertory of atpD, tuf and recA
sequences are also an object of the present invention. The amino acid sequence
data
will be particularly useful for homology modeling of three-dimensional (3D)
structure of the elongation factor Tu, elongation factor G, elongation factor
1 a,
ATPase subunit beta and RecA recombinase. For all these proteins, at least one
structure model has been published using X-ray diffraction data from crystals.
Based
on those structural informations it is possible to use computer sofware to
build 3D
model structures for any other protein having peptide sequence homologies with
the
known structure (Green 1991, Methods in Enzymology, 202:239-252, Taylor, 1994,
Sali, 1995, Curr. Opin. Biotechnol. 6:437-451, Sanchez and Sali, 1997, Curr.
Opin.
Struct. Biol. 7:206-214, Fischer and Eisenberg, 1999, Curr. Opin. Struct.
Biol. 9:208-
211, Guex et al., 1999, Trends Biochem. Sci. 24: 364-367). Model structures of
target
proteins are used for the design or to predict the behavior of ligands and
inhibitors
CA 02307010 2000-OS-19
such as antibiotics. Since EF-Tu and EF-G are already known as antibiotic
targets
(see above) and since the beta subunit of ATPase and RecA recombinase are
essential
to the survival of the microbial cells in natural conditions of infection, all
four
proteins could be considered antibiotic targets. Sequence data, especially the
new
data generated by us could be very useful to assist the creation of new
antibiotic
molecules with desired spectrum of activity. In addition, model structures
could be
used to improve protein function for commercial purposes such as improving
antibiotic production by microbial strains or increasing biomass.
31
CA 02307010 2000-OS-19
BRIEF SUMMARY OF THE INVENTION
Three highly conserved genes, encoding translation elongation factor Tu, the
catalytic
subunit of proton-translocating ATPase and the RecA recombinase, are used to
generate species-specific, genus-specific, group-specific and universal
nucleic acid
probes and amplification primers to rapidly detect and identify bacterial,
fungal and
parasitical pathogens from clinical specimens for diagnosis. The concomittant
detection of associated antibiotic resistance and toxin genes are also under
the scope
of the present invention.
DESCRIPTION OF THE DRAWINGS
Figures 1 and 2 illustrate the principal subdivisions of the tuf and atpD
sequences
repertories, respectively. For the design of primers and probes, depending on
the
needs, one may want to use the complete data set illustrated on the top of the
pyramid
or use only a subset illustrated by the different branching points. Smaller
subdivisions, representing groups, families, genus and species, could even be
made to
extend to the bottom of the pyramid. Because the tuf and atpD sequences are
highly
conserved and evolved with each species, the design of primers and probes does
not
need to include all the sequences within the database or its subdivisions. As
illustrated, in Annexes IV to XX, depending on the use, sequences from a
limited
number of species can be carefully selected to represent: i) only the main
phylogenetic branches from which the intended probes and primers need to be
differentiating, and ii) only the species for which they need to be matching.
However,
for ubiquity purposes, and especially for primers and probes identifying large
groups
of species (genus, family, group or universal, or sequencing primers), the
more data is
included into the sequence analysis, the better the probes and primers will be
suitable
for each particular intended use. Similarly, for specificity purposes, a
larger data set
(or repertory) ensures optimal primers and probes design by reducing the
chance of
employing nonspecific oligonucleotides.
Figure 3 illustrates the approach used to design specific amplification
primers from
fusA as well as from the region between the end of fusA and the beginning of
tuf in
the streptomycin (str) operon.
EXAMPLES AND ANNEXES
The following examples and annexes are intended to be illustrative of the
various methods and compounds of the invention, rather than limiting the scope
thereof.
32
CA 02307010 2000-OS-19
The various annexes show the strategies used for the selection of DNA
amplification primers, nucleic acid hybridization probes or molecular beacon
internal
probes from tuf, atpD, recA, speA, stx, van, pbp, mecA, hexA, pcp, ddl, mtl or
unknown gene sequences:
(i) Annex I shows the amplification primers used for tuf sequences.
(ii) Annex II shows the amplification primers used for atpD sequences.
(iii) Annex III shows the internal probes for nucleic acid hybridization and
specific detection of tuf sequences.
(iv) Annex IV illustrates the strategy used for the selection of the
amplification
primers specific for atpD sequences of the F-type.
(v) Annex V illustrates the strategy used for the selection of the
amplification
primers specific for atpD sequences of the V-type.
(vi) Annex VI illustrates the strategy used for the selection of the
amplification
primers specific for the tuf sequences of organelle lineage (M, the letter M
is
used to indicate that in most cases, the organelle is the mitochondria).
(vii) Annex VII illustrates the strategy used for the selection of the
amplification
primers specific for the tuf sequences of eukaryotes (EF-1).
(viii) Annex VIII illustrates the strategy for the selection of Streptococcus
agalactiae-specific amplification primers from tuf sequences.
(ix) Annex IX illustrates the strategy for the selection of Streptococcus
agalactiae-specific hybridization probes from tuf sequences.
(x) Annex X illustrates the strategy for the selection of Streptococcus
agalactiae-specific amplification primers from atpD sequences.
(xi) Annex XI illustrates the strategy for the selection from tuf sequences of
Candida albicansldubliniensis-specific amplification primers, Candida
albicans-specific hybridization probe and Candida dubliniensis-specific
hybridization probe.
(xii) Annex XII illustrates the strategy for the selection of Staphylococcus-
specific amplification primers from tuf sequences.
(xiii) Annex XIII illustrates the strategy for the selection of the
Staphylococcus
genus-specific hybridization probe from tuf sequences.
(xiv) Annex XIV illustrates the strategy for the selection of Staphylococcus
saprophyticus-specific and Staphylococcus haemolyticus-specific
hybridization probes from tuf sequences.
(xv) Annex XV illustrates the strategy for the selection of Staphylococcus
aureus-specific and Staphylococcus epidermidis-specific hybridization
probes from tuf sequences.
(xvi) Annex XVI illustrates the strategy for the selection of the
Staphylococcus
hominis-specific hybridization probe from tuf sequences.
33
CA 02307010 2000-OS-19
(xvii) Annex XVII illustrates the strategy for the selection of the
Enterococcus
genus-specific amplification primers from tuf sequences.
(xviii) Annex XVIII illustrates the strategy for the selection of the
Enterococcus
faecalis-specific hybridization probe, of the Enterococcus
faecium-specific
hybridization probe and of the Enterococcus casseliflavus flavescens-
gallinarum group-specific hybridization probe from tuf sequences.
(xix) Annex XIX illustrates the strategy for the selection of primers
from tuf
sequences for the identification of platelets contaminants.
(xx) Annex XX illustrates the strategy for the selection of the
universal
l0 amplification primers from atpD sequences.
(xxi) Annex XXI illustrates the amplification primers used for nucleic
acid
amplification from recA sequences.
(xxii) Annex XXII shows the specific and ubiquitous primers for nucleic
acid
amplification from speA sequences.
(xxiii) Annex XXIII illustrates the first strategy for the selection
of Streptococcus
pyogenes-specific amplification primers from speA sequences.
(xxiv) Annex XXIV illustrates the second strategy for the selection
of
Streptococcus pyogenes-specific amplification primers from
speA sequences.
(xxv) Annex XXV illustrates the strategy for the selection of
Streptococcus
pyogenes-specific amplification primers from tuf sequences.
(xxvi) Annex XXVI illustrates the strategy for the selection of Shiga
toxin-
producing, Escherichia coli-specific amplification primers
and hybridization
probe from stx~ sequences.
(xxvii) Annex XXVII illustrates the strategy for the selection of Shiga
toxin-
producing, Escherichia coli-specific amplification primers
and hybridization
probe from stxZ sequences.
(xxviii)
Annex XXVIII
illustrates
the strategy
for the
selection
of vanA-specific
amplification primers from van sequences.
(xxix) Annex XXIX illustrates the strategy for the selection of vanB-
specific
amplification primers from van sequences.
(xxx) Annex XXX illustrates the strategy for the selection of vanC-
specific
amplification primers from vanC sequences.
(xxxi) Annex XXXI illustrates the strategy for the selection of
Streptococcus
pneumoniae-specific amplification primers and hybridization
probes from
pbpl a sequences.
(xxxii) Annex XXXII shows the specific and ubiquitous primers for nucleic
acid
amplification from stx sequences.
(xxxiii ) Annex XXXIII shows the molecular beacon internal probes for
hybridization
and specific detection of toxin sequences.
(xxxiv ) Annex XXXIV shows the specific and ubiquitous primers for
nucleic acid
34
CA 02307010 2000-OS-19
amplification from van sequences.
(xxxv) Annex ~!:XXV shows the internal probes for nucleic acid
hybridization
and
specific detection of van sequences.
(xxxvi) Annex XXXVI shows the specific and ubiquitous primers for
nucleic acid
amplification from pbp sequences.
(xxxvii) Annex XXXVII shows the internal probes for nucleic acid
hybridization
and
specific detection of pbp sequences.
(xxxviii)Annex
XXXVIII
illustrates
the strategy
for the
selection
of vanAB-specific
amplification primers and specific hybridization probes vanA
and vanB from
van sequences.
(xxxix) Annex XXXIX shows the internal probe for nucleic acid hybridization
and
specific detection of mecA.
(xl) Annex XL shows the specific and ubiquitous primers for nucleic
acid
amplification from hexA sequences.
(xli) Annex XLI shows the internal probe for nucleic acid hybridization
and
specific detection of hexA.
(xlii) Annex XLII illustrates the strategy for the selection of
Streptococcus
pneumoniae species-specific amplification primers and hybridization
probe
from hexA sequences.
(xliii) Annex XLIII shows the specific and ubiquitous primers for
nucleic acid
amplification from pcp sequences.
(xliv) Annex XLIV shows the specific and ubiquitous primers for nucleic
acid
amplification from unknown S. saprophyticus gene sequences.
(xlv) Annex XLV shows the molecular beacon internal probes for
hybridization
and specific detection of antibiotic resistance gene sequences.
(xlvi) Annex XLVI shows the molecular beacon internal probe for
hybridization
and specific detection of an unknown S. aureus gene sequence.
(xlvii) Annex XLVII shows the molecular beacon internal probe for
hybridization
and specific detection of tuf sequences.
(xlviii)Annex XLVIII shows the molecular beacon internal probes for
hybridization
and specific detection of ddl and mtl gene sequences.
(xlix) Annex XLIX shows the internal probe for nucleic acid hybridization
and
specific detection of the unknown S. aureus gene.
As shown in these annexes, the selected amplification primers may contain
inosines and/or degenerescences. Inosine is a nucleotide analog able to
specifically
bind to any of the four nucleotides A, C, G or T. Alternatively, degenerated
oligonucleotides which consist of an oligonucleotide mix having two or more of
the
four nucleotides A, C, G or T at the site of mismatches were used. The
inclusion of
inosine andlor of degenerescences in the amplification primers allows mismatch
CA 02307010 2000-OS-19
tolerance thereby permitting the amplification of a wider array of target
nucleotide
sequences (Dieffenbach and Dveksler, 1995 PCR Primer: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Plainview, New York).
EXAMPLES
EXAMPLE 1:
to
Sequencing of bacterial a~D (F-ty,~e and V-type) gene fragments. As shown in
Annex IV, the comparison of publicly available atpD (F-type) sequences from a
variety of bacterial species revealed conserved regions allowing the design of
PCR
primers able to amplify atpD sequences (F-type) from a wide range of bacterial
species. Using primers pairs SEQ ID NOs. 566 and 567, 566 and 814, 568 and
567,
570 and 567, 572 and 567, 569 and 567, 571 and 567, and 700 and 567, it was
possible to amplify and sequence atpD sequences SEQ ID NOs. 242-270, 272-398,
673-674, 737-767, 866-867 and 942-955.
Similarly, Annex V shows how were designed the PCR primers able to amplify
atpD
sequences of the V-type from a wide range of bacterial species. Using primers
SEQ
ID NOs. 681-683, it was possible to amplify and sequence atpD sequences SEQ ID
Nos. 827-832, 929-931, 958 and 966. As the gene was difficult to amplify for
several
species, additional amplification primers were designed inside the original
amplicon
(SEQ ID NOs.1203-1207).
EXAMPLE 2:
Seduencing of eukaryotic atpD (F-type and V-type) gene fragments. The
comparison
3o of publicly available atpD (F-type) sequences from a variety of fungal and
parasitical
species revealed conserved regions allowing the design of PCR primers able to
amplify atpD sequences from a wide range of fungal and parasitical species.
Using
primers pairs SEQ ID NOs. 568 and 573, 574 and 573, 574 and 708, and 566 and
567, it was possible to amplify and sequence atpD sequences SEQ ID NOs. 458-
497,
530-538, 663, 667, 676, 678-680, 768-778, 856-862, 889-896 and 941.
In the same manner, the primers described in Annex V (SEQ ID NOs. 681-
683)could
amplify the atpD (V-type) gene from fungal and parasitical species. We were
thus
able to sequence SEQ ID Nos. 834-839, 956-957, and 959-965.
EXAMPLE 3:
Sequencing of eukar~otic tuf (EF-1) eng a fragments. As shown in Annex VII,
the
comparison of publicly available tuf (EF-1) sequences from a variety of fungal
and
36
CA 02307010 2000-OS-19
parasitical species revealed conserved regions allowing the design of PCR
primers
able to amplify tuf sequences from a wide range of fungal and parasitical
species.
Using primers pairs SEQ ID NOs. 558 and 559, 813 and 559, 558 and 815, 560 and
559, 653 and 559, 558 and 655, and 654 and 559, it was possible to amplify and
sequence tuf sequences SEQ ID NOs. 399-457, 509-529, 622-624, 677, 779-790,
840-842, 865, and 897-903.
EXAMPLE 4:
Sec~uencin~of eukar~otic turf (organelle origin, M) gene fragments. As shown
in
Annex VI, the comparison of publicly available tuf (organelle origin, M)
sequences
from a variety of fungal and parasitical organelles revealed conserved regions
allowing the design of PCR primers able to amplify tuf sequences of several
organelles belonging to a wide range fungal and parasitical species. Using
primers
pairs SEQ ID NOs. 664 and 652, 664 and 561, 911 and 914, 912 and 914, 913 and
915, 916 and 561, and 664 and 917, it was possible to amplify and sequence tuf
sequences SEQ ID NOs. 498-508, 791-792, 843-855, and 904-910.
EXAMPLE 5:
_Specific identification of Streptococcus agalactiae using tuf seguences. As
shown in
Annex VIII, the comparison of tuf sequences from a variety of bacterial
species
allowed the selection of PCR primers specific for S. agalactiae. The strategy
used to
design the PCR primers was based on the analysis of a multiple sequence
alignment
of various tuf sequences. The multiple sequence alignment includes the tuf
sequences
of four bacterial strains from the target species as well as tuf sequences
from other
species and bacterial genera, especially representatives of closely related
species. A
careful analysis of this alignment allowed the selection of oligonucleotide
sequences
which are conserved within the target species but which discriminate sequences
from
other species and genera, especially from the closely related species, thereby
permitting the species-specific and ubiquitous detection and identification of
the
target bacterial species.
The chosen primer pair, oligos SEQ ID NO. 549 and SEQ ID NO. 550, gives an
amplification product of 252 bp. Standard PCR was carned out using 0.4 ~.M of
each
primer, 2.5 mM MgCl2, BSA 0.05 mM, 1X Taq Buffer (Promega), dNTP 0.2 mM
(Pharmacia), 1 ~,1 Taq DNA polymerase (Promega) 0.025 U/,ul combined with
TaqStart 5 ng/~,1 (Clontech Laboratories Inc., Palo Alto), 1 ~,l of genomic
DNA
sample in a final volume of 20 ~,l using a PTC-200 thermocycler (MJ Research
Inc.).
The optimal cycling conditions for maximum sensitivity and specificity were 3
minutes at 95 °C for initial denaturation, then forty cycles of two
steps consisting of 1
second at 95 °C and 30 seconds at 62 °C, followed by terminal
extension at 72 °C for
37
CA 02307010 2000-OS-19
2 minutes. Amplification was monitored on agarose gel electrophoresis by
staining
the DNA with ethidium bromide.
Specificity of the assay was tested by adding into the PCR reactions, 0.1 ng
of
genomic DNA from each of the bacterial species listed in Table 8. Strong
amplification was observed only for the 5 S. agalactiae strains listed. Of the
other
bacterial species, including 32 species representative of the vaginal flora
and 27 other
streptococcal species, only S. acidominimus yielded amplification. The signal
for 0.1
ng of S. acidominimus genomic DNA was weak and the detection limit for this
species was 10 pg (corresponding to more than 4000 genome copies) while the
detection limit for S. agalactiae was 2.5 fg (corresponding to one genome
copy) of
genomic DNA.
To increase the specificity of the assay, internal probes were designed for
FRET
(Fluorescence Resonance Energy Transfer) detection using the LightCycler
(Idaho
Technology). As illustrated in Annex IX, a multiple sequence alignment of
streptococcal tuf sequence fragments corresponding to the 252 by region
amplified by
primers SEQ ID NO. 549 and SEQ ID NO. 550, was used for the design of internal
probes TSagHF436 (SEQ ID NO. 582) and TSagHF465 (SEQ ID NO. 583). The
region of the amplicon selected for internal probes contained sequences unique
and
specific to S. agalactiae. SEQ ID NO. 583, the more specific probe, is
labelled with
fluorescein in 3', while SEQ ID NO. 582, the less discriminant probe, is
labelled with
CY5 in 5' and blocked in 3' with a phosphate group. However, since the FRET
signal
is only emitted if both probes are adjacently hybridized on the same target
amplicon,
detection is highly specific.
Real-time detection of PCR products using the LightCyclerTM was carried out
using
0.4 ~,M of each primer (SEQ ID NO. 549-550), 0.2 ~,M of each probe (SEQ ID NO.
582-583), 2.5 mM MgCl2, BSA 450 ~,g/ml, 1X PC2 Buffer (AB Peptides, St-Louis,
3o MO), dNTP 0.2 mM (Pharmacia), KlenTaqlTM DNA polymerase 0.5 U (AB
Peptides) 0.025 U/~.l combined with TaqStart (Clontech Laboratories Inc., Palo
Alto),
0.7 ~,1 of genomic DNA sample in a final volume of 7 ~,l using a LightCycler
thermocycler (Idaho Technology). The optimal cycling conditions for maximum
sensitivity and specificity were 3 minutes at 94 °C for initial
denaturation, then forty
cycles of three steps consisting of 0 second (this setting meaning the
LightCycler will
reach the target temperature and stay at it for its minimal amount of time) at
94 °C,
10 seconds at 64 °C, 20 seconds at 72 °C. Amplification was
monitored during each
annealing steps using the fluorescence ratio. The streptococcal species having
close
sequence homologies with the tuf sequence of S. agalactiae (S. acidominimus,
S.
anginosus, S. bovis, S. dysgalactiae, S. equi, S. ferns, S. gordonii, S.
intermedius, S.
38
CA 02307010 2000-OS-19
parasanguis, S. parauberis, S. salivarius, S. sanguis, S. suis, and of course
S.
agalactiae) were tested in the LightCycler with 0.07 ng of genomic DNA per
reaction. This time, only S. agalactiae yielded an amplification signal, hence
demonstrating that the assay is species-specific. With the LightCycler assay
using the
internal FRET probes, the detection limit for S. agalactiae was 12.5 fg
(corresponding to five genome copies) of genomic DNA.
EXAMPLE 6:
Specific identification of Streptococcus a~alactiae using atpD seauences. As
shown
in Annex XIV, the comparison of atpD sequences from a variety of bacterial
species
allowed the selection of PCR primers specific for S. agalactiae. The primer
design
strategy is similar to the strategy described in the preceding example except
that atpD
sequences were used in the alignment (see Annex X).
Four primers were selected, ASag42 (SEQ ID NO. 627), ASag52 (SEQ ID NO. 628),
ASag206 (SEQ ID NO. 625) and ASag371 (SEQ ID NO. 626). The following
combinations of these four primers give four amplicons; SEQ ID NO. 627 + SEQ
ID
NO. 625 = 190 bp, SEQ ID NO. 628 + SEQ ID NO. 625 = 180 bp, SEQ ID NO. 627
+ SEQ ID NO. 626 = 355 bp, and SEQ ID NO. 628 + SEQ ID NO. 626 = 345 bp.
Standard PCR was carried out on PTC-200 thermocyclers (MJ Research Inc) using
0.4 ~,M of each primers pair, 2.5 mM MgCl2, BSA 0.05 mM, 1X taq Buffer
(Promega), dNTP 0.2 mM (Pharmacia), 1 ~,1 taq DNA polymerase (Promega) 0.025
U/~,1 combined with TaqStart 5 ng/~,1 (Clontech Laboratories Inc., Palo Alto),
1 ~.1 of
genomic DNA sample in a final volume of 20 ~,1 using a PTC-200 thermocycler
(MJ
Research Inc.). The optimal cycling conditions for maximum sensitivity and
specificity were adjusted for each primer pair. Three minutes at 95 °C
for initial
denaturation, then forty cycles of two steps consisting of 1 second at 95
°C and 30
seconds at the optimal temperature specified below were followed by terminal
extension at 72 °C for 2 minutes. Amplification was monitored on
agarose gel
electrophoresis by staining the DNA with ethidium bromide. Since atpD
sequences
are relatively more specific than tuf sequences, only the more closely related
species
namely, the steptococcal species listed in table 9, were tested.
All four primer pairs only amplified the six S. agalactiae strains. With an
annealing
temperature of 63 °C, the primer pair SEQ ID NO. 627 + SEQ ID NO. 625
had a
sensitivity of 1-5 fg (equivalent to 1-2 genome copies). At 55 °C, the
primer pair SEQ
ID NO. 628 + SEQ ID NO. 625 had a sensitivity of 2.5 fg (equivalent to 1
genome
copy). At 60 °C, the primer pair SEQ ID NO. 627 + SEQ ID NO. 626 had a
39
CA 02307010 2000-OS-19
sensitivity of 10 fg (equivalent to 4 genome copies). At 58 °C, the
primer pair SEQ
ID NO. 628 + SEQ ID NO. 626 had a sensitivity of 2.5-5 fg (equivalent to 1-2
genome copies). This proves that all four primer pairs can detect S.
agalactiae with
high specificity and sensitivity. Together with example 5, this example
demonstrates
that both tuf and atpD sequences are suitable targets for the identification
of
microorganisms at the species level.
EXAMPLE 7:
l0 Development of a PCR assay for detection and identification of
staphylococci at
genus and species levels.
Materials and Methods
Bacterial strains. The specificity of the PCR assay was verified by using a
panel of
ATCC (America Type Culture Collection) and DSMZ (Deutsche Sammlung yon
Mikroorganismen and Zellkulturen GmbH ; German Collection of Microorganisms
and Cell Cultures) reference strains consisting of 33 gram-negative and 47
gram-
positive bacterial species (Table 12). In addition, 295 clinical isolates
representing 11
different species of staphylococci from the microbiology laboratory of the
Centre
Hospitalier Universitaire de Quebec, Pavilion Centre Hospitalier de
1'Universite
Laval (CHUL) (Ste-Foy, Quebec, Canada) were also tested to further validate
the
Staphylococcus-specific PCR assay. These strains were all identified by using
(i)
conventional methods or (ii) the automated MicroScan Autoscan-4 system
equipped
with the Positive BP Combo Panel Type 6 (Dade Diagnostics, Mississauga,
Ontario,
Canada). Bacterial strains from frozen stocks kept at -80 °C in brain
heart infusion
(BHI) broth containing 10% glycerol were cultured on sheep blood agar or in
BHI
broth (Quelab Laboratories Inc, Montreal, Quebec, Canada).
PCR primers and internal probes. Based on multiple sequence alignments,
regions
of the tuf gene unique to staphylococci were identified. Staphylococcus genus-
specific
PCR primers TStaG422 (SEQ ID NO. 553) and TStaG765 (SEQ ID NO. 575) were
derived from these regions (Annex XII). These PCR primers are displaced by two
nucleotide positions compared to original Staphylococcus genus-specific PCR
primers
described in previous patent application W098/20157 (SEQ ID NOs. 17 and 20 in
the
said patent application). These modifications were done to ensure specificity
and
ubiquity of the primer pair, in the light of new tuf sequence data revealed in
the present
patent application for several additional streptococcal species and strains.
Similarly, sequence alignments analysis were performed to design genus and
species
CA 02307010 2000-OS-19
specific internal probes (see Annexes XIII to XVI). Two internal probes for
Staphylococcus-genus (SEQ ID NOs. 605-606), five for S. aureus (SEQ ID NOs.
584-
588), five for S. epidermidis (SEQ ID NO. 589-593), two for S. haemolyticus
(SEQ ID
NOs. 594-595), three for S. hominis (SEQ ID NOs. 596-598), four for S.
saprophyticus
(SEQ ID NOs. 599-601 and 695), and two for coagulase-negative Staphylococcus
species S. epidermidis, S. hominis, S. saprophyticus, S. auricularis, S.
capitis, S.
haemolyticus, S. lugdunensis, S. simulans, S. cohnii and S. warneri (SEQ ID
NOs.
1175-1176) were designed. The range of mismatches between the Staphylococcus-
specific 371-by amplicon and each of the 20-mer species-specific internal
probes was
from 1 to 5, in the middle of the probe when possible. No mismatches were
present in
the two Staphylococcus-genus probes for the 11 species analyzed : S. aureus,
S.
auricularis, S. capitis, S. cohnii, S. epidermidis, S. haemolyticus, S.
hominis, S.
lugdunensis, S. saprophyticus, S. simulans and S. warneri. In order to verify
the intra-
specific sequence conservation of the nucleotide sequence, sequences were
obtained for
the 371-by amplicon from five unrelated ATCC and clinical strains for each of
the
species S. aureus, S. epidermidis, S. haemolyticus, S. hominis and S.
saprophyticus. The
OligoTM (version 5.0) primer analysis software (National Biosciences,
Plymouth,
Minn.) was used to confirm the absence of self-complementary regions within
and
between the primers or probes. When required, the primers contained inosines
or
degenerated nucleotides at one or more variable positions. Oligonucleotide
primers and
probes were synthesized on a model 394 DNA synthesizer (Perkin-Elmer Corp.,
Applied Biosystems Division, Mississauga, Ontario, Canada). Detection of the
hybridization was performed with the DIG-labeled dUTP incorporated during the
amplification with the Staphylococcus-specific PCR assay, and the
hybridization signal
was detected with a luminometer (Dynex Technologies) as described above in the
section on luminescent detection of amplification products. Annexes XIII to
XVI
illustrate the strategy for the selection of several internal probes.
PCR amplification. For all bacterial species, amplification was performed from
purified genomic DNA or from a bacterial suspension whose turbidity was
adjusted to
that of a 0.5 McFarland standard, which corresponds to approximately 1.5 x 108
bacteria per ml. One nanogram of genomic DNA or 1 ~1 of the standardized
bacterial
suspension was transferred directly to a 19 ~1 PCR mixture. Each PCR reaction
contained 50 mM KCI, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2,
0.2 ~.M (each) of the two Staphylococcus genus-specific primers (SEQ ID NOs.
553
and 575), 200 ~M (each) of the four deoxynucleoside triphosphates (Pharmacia
Biotech), 3.3 ~ug/~1 bovine serum albumin (BSA) (Sigma-Aldrich Canada Ltd,
Oakville,
Ontario, Canada), and 0.5 U Taq polymerase (Promega) coupled with TaqStart~
Antibody (Clontech). The PCR amplification was performed as follows : 3 min.
at 94
41
CA 02307010 2000-OS-19
°C for initial denaturation, then forty cycles of two steps consisting
of 1 second at 95 °C
and 30 seconds at 55 °C, plus a terminal extension at 72 °C for
2 minutes.
Amplification was monitored on agarose gel electrophoresis by staining the
amplified
DNA with ethidium bromide.
For determination of the sensitivities of the PCR assays, two-fold dilutions
of
purified genomic DNA were used to determine the minimal number of genome
copies
which can be detected.
l0 Results
Amplifications with the Staphylococcus genus-specific PCR assay. The
specificity
of the assay was assessed by performing 30-cycle and 40-cycle PCR
amplifications
with the panel of gram-positive (47 species from 8 genera) and gram-negative
(33
species from 22 genera) bacterial species listed in Table 12. The PCR assay
was able to
detect 27 of 27 staphylococcal species tested in both 30-cycle and 40-cycle
regimens.
For 30-cycle PCR, all bacterial species tested other than staphylococci were
negative.
For 40-cycle PCR, Enterococcus faecalis, Lactobacillus acidophilus,
Lactococcus
lactis, Macrococcus caseolyticus, Streptocuccus agalactiae and S. mutans were
slightly
positive for the Staphylococcus-specific PCR assay. The other species tested
remained
negative. Ubiquity tests performed on a collection of 295 clinical isolates
provided by
the microbiology laboratory of the Centre Hospitalier Universitaire de Quebec,
Pavillon Centre Hospitalier de 1'Universite Laval (CHUL), including
Staphylococcus
aureus (n=34), S. auricularis (n=2), S. capitis (n=19), S. cohnii (n=5), S.
epidermidis
(n=18), S. haemolyticus (n=21), S. hominis (n=73), S. lugdunensis (n=17), _ S.
saprophyticus (n=6), S. simulans (n=3), S. warneri (n=32) and Staphylococcus
sp.
(n=65), showed a uniform amplification signal with the 30-cycle PCR assays and
a
perfect relation between the genotype and classical identification schemes.
The sensitivity of the Staphylococcus-specific assay with 30-cycle and 40-
cycle PCR
protocols was determined by using purified genomic DNA from the 11
staphylococcal
species previously mentioned. For PCR with 30 cycles, a detection limit of 50
copies of
genomic DNA was consistently obtained. In order to enhance the sensitivity of
the
assay, the number of cycles was increased. For 40-cycle PCR assays, the
detection limit
was lowered to a range of 5-10 genome copies, depending on the staphylococcal
species tested.
Hybridization between the Staphylococcus-specific 371-by amplicon and species-
specific or genus-specific internal probes. Inter-species polymorphism was
4o sufficient to generate species-specific internal probes for each of the
principal
42
CA 02307010 2000-OS-19
species involved in human diseases (S. aureus, S. epidermidis, S.
haemolyticus, S.
hominis and S. saprophyticus). In order to verify the intra-species sequence
conservation of the nucleotide sequence, sequences comparisons were performed
on
the 371-by amplicon from five unrelated ATCC and clinical strains for each of
5
staphylococcal species: S. aureus, S. epidermidis, S. haemolyticus, S. hominis
and S.
saprophyticus. Results showed a high level of conservation of nucleotide
sequence
between different unrelated strains from the same species. This sequence
information
allowed the development of staphylococcal species identification assays using
species-specific internal probes hybridizing to the 371-by amplicon. These
assays are
specific and ubiquitous for those five staphylococcal species. In addition to
the
species-specific internal probes, the genus-specific internals probes were
able to
recognize most Staphylococcus species.
EXAMPLE 8:
Differentiating between the two closely related yeast species Candida albicans
and
Candida dubliniensis. It is often useful for the clinician to be able to
differentiate
between two very closely related species of microorganisms. Candida albicans
is the
2o most important cause of invasive human mycose. In the recent years, a very
closely
related species, Candida dubliniensis, was isolated in immunosuppressed
patients.
These two species are difficult to distinguish by classic biochemical methods.
This
example demonstrates the use of tuf sequences to differentiate Candida
albicans and
Candida dubliniensis. PCR primers TCa1528 and TCa1676 were selected for their
ability to specifically amplify a tuf (elongation factor 1 alpha type)
fragment from
both species (see Annex XI for primer positions and previous patent
application
W098/20157 for SEQ ID NOs. 11-12). Within this tuf fragment, a region
differentiating C. albicans and C. dubliniensis by two nucleotides was
selected and
used to design two internal probes (see Annex XI for probe design, SEQ ID NOs.
577
and 578) specific for each species. Amplification of genomic DNA from C.
albicans
and C. dubliniensis was carried out using DLG-11-dUTP as described above in
the
section on chemiluminescent detection of amplification products. Internal
probes
SEQ ID NOs. 577 and 578 were immobilized on the bottom of individual
microtiter
plates and hybridization was carried out as described above in the above
section on
luminescent detection of amplification products. Luminometer data showed that
the
amplicon from C. albicans hybridized only to probe SEQ ID NO. 577 while the
amplicon from C. dubliniensis hybridized only to probe SEQ ID NO. 578, thereby
demonstrating that each probe was species-specific.
4o EXAMPLE 9:
43
CA 02307010 2000-OS-19
_Specific identification of Entamoeba histolytica. Upon analysis of tuf
(elongation
factor 1 alpha) sequence data, it was possible to find four regions where
Entamoeba
histolytica sequences remained conserved while other parasitical and
eukaryotic
species have diverged. Primers TEntG38 (SEQ ID NO. 703), TEntG442 (SEQ ID
NO. 704), TEntG534 (SEQ ID NO. 705), and TEntG768 (SEQ ID NO. 706) were
designed so that SEQ ID NO. 703 could be paired with the three other primers.
On
PTC-200 thermocyclers (MJ Research), the cycling conditions for initial
sensitivity
and specificity testing were 3 min. at 94 °C for initial denaturation,
then forty cycles
of two steps consisting of 1 second at 95 °C and 30 seconds at 55
°C, followed by
terminal extension at 72 °C for 2 minutes. Amplification was monitored
on agarose
gel electrophoresis by staining the amplified DNA with ethidium bromide. The
three
primer pairs could detect the equivalent of less than 200 E. histolytica
genome copies.
Specificity was tested using 0.5 ng of purified genomic DNA from a panel of
microorganisms including Babesia bovis, Babesia microtti, Candida albicans,
Crithidia fasciculata, Leishmania major, Leishmania hertigi and Neospora
caninum.
Only E. histolytica DNA could be amplified, thereby suggesting that the assay
was
species-specific.
2o EXAMPLE 10:
Sensitive identification of Chlamydia trachomatis. Upon analysis of tuf
sequence
data, it was possible to find two regions where Chlamydia trachomatis
sequences
remained conserved while other species have diverged. Primers Ctr82 (SEQ ID
NO.
554) and Ctr249 (SEQ ID NO. 555) were designed. With the PTC-200 thermocyclers
(MJ Research), the optimal cycling conditions for maximum sensitivity and
specificity were determined to be 3 min. at 94 °C for initial
denaturation, then forty
cycles of two steps consisting of 1 second at 95 °C and 30 seconds at
60 °C, followed
by terminal extension at 72 °C for 2 minutes. Amplification was
monitored on
agarose gel electrophoresis by staining the amplified DNA with ethidium
bromide.
The assay could detect the equivalent of 8 C. trachomatis genome copies.
Specificity
was tested on 0.1 ng of purified genomic DNA from a panel of microorganisms
including 22 species commonly encountered in the vaginal flora (Bacillus
subtilis,
Bacteroides fragilis, Candida albicans, Clostridium difficile, Corynebacterium
cervicis, Corynebacterium urealyticum, Enterococcus faecalis, Enterococcus
faecium, Escherichia coli, Fusobacterium nucleatum, Gardnerella vaginalis,
Haemophilus influenzae, Klebsiella oxytoca, Lactobacillus acidophilus,
Peptococcus
niger, Peptostreptococcus prevotii, Porphyromonas asaccharolytica, Prevotella
melaninogenica, Propionibacterium acnes, Staphylococcus aureus, Streptococcus
acidominimus, and Streptococcus agalactiae). Only C. trachomatis DNA could be
44
CA 02307010 2000-OS-19
amplified, thereby suggesting that the assay was species-specific.
EXAMPLE 11:
Genus-specific identification of enterococci. Upon analysis of tuf sequence
data and
comparison with the repertory of tuf sequences, it was possible to find two
regions
where Enterococcus sequences remained conserved while other genera have
diverged
(Annex XVII). Primer pair Encg313dF and Encg599c (SEQ ID NOs. 1137 and 1136)
was tested for its specificity by using purified genomic DNA from a panel of
bacteria
listed in Table 10. Using the PTC-200 thermocycler (MJ Research), the optimal
cycling conditions for maximum sensitivity and specificity were determined to
be 3
min. at 94 °C for initial denaturation, then forty cycles of two steps
consisting of 1
second at 95 °C and 30 seconds at 55 °C, followed by terminal
extension at 72 °C for
2 minutes. Amplification was monitored on agarose gel electrophoresis by
staining
the amplified DNA with ethidium bromide. The 18 enterococcal species listed in
Table 10 were all amplified efficiently. The only other species amplified were
Abiotrophia adiacens, Gemella haemolysans and Gemella morbillorum, three gram-
positive species. Sensitivity tested with several strains of E. casseliflavus,
E. faecium,
E. faecalis, E. flavescens and E. gallinarum and with one strain of each other
2o Enterococcus species listed in Table 10 ranged from 1 to 10 genome copies.
The
sequence variation within the 308-by amplicon was sufficient so that nested
PCR or
internal probes could be used to speciate the amplicon and differenciate
enterococci
from Abiotrophia adiacens, Gemella haemolysans and Gemella morbillorum,
thereby
allowing to achieve excellent specificity. Species-specific internal probes
were
generated for each of the clinically important species, E. faecalis (SEQ ID
NO.
1174), E. faecium (SEQ ID NO. 602), and the E. casseliflavus, E. flavescens,
E.
gallinarum group (SEQ ID NO. 1122) (Annex XVIII). The species-specific
internal
probes were able to differentiate their respective Enterococcus species from
all other
Enterococcus species. These assays are sensitive, specific and ubiquitous for
those
five Enterococcus species.
EXAMPLE 12:
Identification of the major bacterial platelets contaminants using tuf
seguences in a
multiplex test. Blood platelets preparations need to be monitored for
bacterial
contaminations. The tuf sequences of 17 important bacterial contaminants of
platelets
were aligned. As shown in Annex XIX, analysis of these sequences allowed the
design PCR primers. Since in the case of contamination of platelet
concentrates,
detecting all species (not just the more frequently encountered ones) is
desirable,
perfect specificity of primers was not an issue in the design. However,
sensitivity is
CA 02307010 2000-OS-19
important. That is why, to avoid having to put too much degeneracy, only the
most
frequent contaminants were included in primer design, knowing that the
selected
primers would anyway be able to amplify more species than the 17 used in the
design. Oligonucleotide sequences which are conserved in these 17 major
bacterial
contaminants of platelet concentrates were chosen (oligos Tplaq 769 and Tplaq
991,
respectively SEQ ID NOs. 636 and 637) thereby permitting the detection of
these
bacterial species. However, sensitivity was slightly deficient with
staphylococci. To
ensure maximal sensitivity in the detection of all the more frequent bacterial
contaminants, a multiplex assay also including oligonucleotide primers
targetting the
l0 Staphylococcus genera (oligos Stag 422, SEQ ID NO. 553; and Stag 765, SEQ
ID
NO. 575) was developed. The bacterial species detected with the assay are
listed in
Table 14.
The primer pairs, oligos SEQ ID NO. 636 and SEQ ID NO. 637 that give an
amplification product of 245 pb, and oligos SEQ ID NO. 553 and SEQ ID NO. 575
that give an amplification product of 368 pb, were used simultaneously in the
multiplex PCR assay. Real-time detection of these PCR products was made on the
LightCycler thermocycler (Idaho Technology) using SYBR~ Green I (Molecular
Probe Inc.). SYBR~ Green I is a fluorescent dye that binds specifically to
double-
stranded DNA. It thus binds to DNA products as they are synthesized. The
measure
of SYBR~ Green I fluorescence at the end of each elongation cycle indicates
the
amount of DNA duplex generated by specific DNA fragment amplification and
primer-dimer formation.
Fluorogenic detection of PCR products with the LightCycler was carried out
using
1.0 mM of both Tplaq primers (SEQ ID NOs. 636-637) and 0.4 mM of both TStaG
primers (SEQ ID NOs. 553 and 575), 2.5 mM MgCl2, BSA 500 mg/ml , dNTP 0.2
mM (Pharmacia), lOX PCR reaction buffer (Boerhinger Mannheim) and taq DNA
polymerase (Boerhinger Mannheim) 0.025 U/ml combined with TaqStart 5 ng/ml
(Clontech), and 0.7 ml of genomic DNA sample in a final volume of 7 ml. The
optimal cycling conditions for maximum sensitivity were 1 minute at 94
°C for initial
denaturation, then forty-five cycles of three steps consisting of 0 second at
95 °C, 5
seconds at 60 °C and 9 seconds at 72 °C. Amplification was
monitored during each
elongation cycle by measuring the level of SYBR~ Green I. However, real
analysis
takes place after PCR. Melting curves are done for each sample and
transformation of
the melting peak allows determination of Tm. Thus primer-dimer and specific
PCR
product are discriminated. With this assay, all prominent bacterial
contaminants of
platelet concentrates listed in Annex XIX and Table 14 were detected.
Sensitivity
tests were performed on the 9 most frequent bacterial contaminants of
platelets. The
detection limit was less than 20 genome copies for E. cloacae, B. cereus, S.
46
CA 02307010 2000-OS-19
choleraesuis and S. marcescens; less than 15 genome copies for P. aeruginosa;
and 2
to 3 copies were detected for S. aureus, S. epidermidis, E. coli and K.
pneumoniae.
Further refinements of assay conditions should increase sensitivity levels.
EXAMPLE 13:
The resolving~power of the tuf and atpD sequences databases is comparable to
the
biochemical methods for bacterial identification. The present gold standard
for
bacterial identification is mainly based on key morphological traits and
batteries of
l0 biochemical tests. Here we demonstrate that the use of tuf and atpD
sequences
combined with simple phylogenetic analysis of databases formed by these
sequences
is comparable to the gold standard. In the process of acquiring data for the
tuf
sequences, we sequenced the tuf gene of a strain that was given to us labelled
as
Staphylococcus hominis ATCC 35982. That tuf sequence (SEQ ID NO. 192) was
incorporated into the tuf sequences database and subjected to a basic
phylogenic
analysis using the Pileup command from version 10 of the GCG package (Genetics
Computer Group, inc.). This analysis indicated that SEQ ID NO. 192 is not
associated with other S. hominis strains but rather with the S. warneri
strains. The
ATCC 35982 strain was sent to the reference laboratory of the Laboratoire de
Sante
publique du Quebec (LSPQ). They used the classic identification scheme for
staphylococci (Kloos and Schleifer, 1975., J. Clip. Microbiol. 1:82-88). Their
results
shown that although the colonial morphology could correspond to S. hominis,
the
more precise biochemical assays did not. These assays included discriminant
mannitol, mannose and ribose acidification tests as well as rapid and dense
growth in
deep thioglycolate agar. The LSPQ report identified strain ATCC 35982 as S.
warneri which confirms our database analysis. The same thing happened for S.
warneri (SEQ ID NO. 187) which had initially been identified as S.
haemolyticus by
a routine clinical laboratory using a low resolving power automated system
(MicroScan, AutoScan-4TM). Again, the tuf and LSPQ analysis agreed on its
identification as S. warneri. In numerous other instances, in the course of
acquiring
tuf and atpD sequence data from various species and genera, analysis of our
tuf
and/or atpD sequence databases permitted the exact identification of
mislabelled or
erroneously identified strains. These results clearly demonstrate the
usefulness and
the high resolving power of our sequence-based identification assays using the
tuf
and atpD sequences databases.
EXAMPLE 14:
Detection of group B stre~,tococci in clinical specimens.
47
CA 02307010 2000-OS-19
Introduction
Streptococcus agalactiae, the group B streptococcus (GBS), is responsible for
a
severe illness affecting neonate infants. The bacterium is passed from the
healthy
carrier mother to the baby during delivery. To prevent this infection, it is
recommended to treat expectant mothers susceptible of carrying GBS in their
anovaginal flora. Carrier status is often a transient condition and rigorous
monitoring
requires cultures and classic bacterial identification weeks before delivery.
To
improve the diagnostic and identification of GBS we developped a rapid,
specific and
l0 sensitive PCR test fast enough to be performed right at delivery.
Materials and Methods
GBS Clinical Specimens. A total of 66 duplicate anovaginal swabs were
collected from 41 consenting pregnant women admitted for delivery at the
Centre
Hospitalier Universitaire de Quebec, Pavillon Saint-Fran~ois d'Assise
following the
CDC recommendations. The samples were obtained either before or after rupture
of
membranes. The swab samples were tested at the Centre de Recherche en
Infectiologie de 1'Universite Laval within 24 hours of collection. Upon
receipt, one
swab was cut and then the tip of the swab was added to GNS selective broth for
identification of group B streptococci (GBS) by the standard culture methods
recommended by the Center for Diseases Control. The other swab was processed
following the instruction of the IDI DNA extraction kit (Infectio Diagnotics
(IDI)
Inc.) prior to PCR amplification.
Oligonucleotides. PCR primers, Tsag340 (SEQ ID NO. 549) and Tsag552
(SEQ ID NO. 550) complementary to the regions of the tuf gene unique for GBS
were designed based upon a multiple sequence alignment using our repertory of
tuf
sequences. Oligo primer analysis software (version 5.0) (National Biosciences)
was
used to analyse primers annealing temperature, secondary structure potential
as well
as mispriming and dimerization potential. The primers were synthesized using a
model 391 DNA synthesizer (Perkin-Elmer).
A pair of fluorescently labeled adjacent hybridization probes Sag465-F (SEQ ID
NO. 583) and Sag436-C (SEQ ID NO. 582) were synthesized and purified by Operon
Technologies. They were designed to meet the recommendations of the
manufacturer
(Idaho Technology) and based upon multiple sequence alignment analysis using
our
repertory of tuf sequences to be specific and ubiquitous for GBS. These
adjacent
probes, which are separated by one nucleotide, allow fluorescence resonance
energy
transfer (FRET), generating an increased fluorescence signal when both
hybridized
simultaneously to their target sequences. The probe SEQ ID NO. 583 was labeled
with FITC in 3 prime while SEQ ID NO. 582 was labeled with Cy5 in 5 prime. The
48
CA 02307010 2000-OS-19
Cy5-labeled probes contained a 3'-blocking phosphate group to prevent
extension of
the probes during the PCR reactions.
PCR Amplification. Conventional amplifications were performed either from 2
~.1 of a purified genomic DNA preparation or cell lysates of anovaginal
specimens.
The 20 ~,1 PCR mixture contained 0.4 ~,M of each GBS-specific primer (SEQ ID
NOs. 549-550), 200 ~uM of each deoxyribonucleotide (Pharmacia Biotech), 10 mM
Tris-HCl (pH 9.0), 50 mM KCI, 0.1% Triton X-100, 2.5 mM MgCl2, 3.3 mg/ml
bovine serum albumin (BSA) (Sigma), and 0.5 U of Taq polymerase (Promega)
combined with the TaqStart antibody (Clontech). The TaqStart antibody, which
is a
neutralizing monoclonal antibody of Taq DNA polymerase, was added to all PCR
reactions to enhance the efficiency of the amplification. The PCR mixtures
were
subjected to thermal cycling (3 min at 95 °C and then 40 cycles of 1 s
at 95 °C, and
30 s at 62 °C with a 2-min final extension at 72 °C) with a PTC-
200 DNA Engine
thermocycler (MJ research). The PCR-amplified reaction mixture was resolved by
agarose gel electrophoresis.
The LightCyclerTM PCR amplifications were performed with 1 ~.1 of the same
preparation as described above. The 101 amplification mixture consisted of 0.4
~,M
each GBS-specific primer (SEQ ID NOs. 549-550), 200 ,uM each dNTP, 0.2 ~,M
each
fluorescently labeled probe (SEQ ID NOs. 582-583), 300 ~.g/ml BSA (Sigma), and
1
~1 of l Ox PC2 buffer (containing 50 mM Tris-HCl (pH 9.1 ), 16 mM ammonium
sulfate, 3.5 mM Mg2+, and 150 ~,g/ml BSA) and 0.5 U KlenTaqlTM (AB Peptides)
coupled with TaqStartTM antibody (Clontech). KlenTaqlTM is a highly active and
more heat-stable DNA polymerase without 5' -exonuclease activity. This
prevents
hydrolysis of hybridized probes by the 5' to 3' exonuclease activity. A volume
of 7 ~ul
of the PCR mixture was transferred into a composite capillary tube (Idaho
Technology). The tubes were then centrifuged to move the reaction mixture to
the
tips of the capillaries and then cleaned with optical-grade methanol.
Subsequently the
capillaries were loaded into the carousel of a LC32 LightCyclerTM (Idaho
Technology), an instrument that combines rapid-cycle PCR with fluorescence
analysis for continuous monitoring during amplification. The PCR reaction
mixtures
were subjected to a denaturation step at 94 °C for 3 min followed by 45
cycles of 0 s
at 94 °C, 20 s at 64 °C and 10 s at 72 °C with a
temperature transition rate of 20 °C/s.
Fluorescence signals were obtained at each cycle by sequentially positioning
each
capillary on the carousel at the focus of optical elements affiliated to the
built-in
fluorimeter for 100 milliseconds. Complete amplification and analysis required
about
35 min.
Specificity And Sensitivity Tests. The specificity of the conventional and
LightCyclerTM PCR assays was verified by using purified genomic DNA (0.1
ng/reaction) from a battery of ATCC reference strains representing 35
clinically
relevant gram-positive species (Abiotrophia defectiva ATCC 49176,
Bifidobacterium
49
CA 02307010 2000-OS-19
breve ATCC 15700, Clostridium difficile ATCC 9689, Corynebacterium urealyticum
ATCC 43042, Enterococcus casseliflavus ATCC 25788, Enterococcus durans ATCC
19432, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 19434,
Enterococcus gallinarum ATCC 49573, Enterococcus raffinosus ATCC 49427,
Lactobacillus reuteri ATCC 23273, Lactococcus lactic ATCC 19435, Listeria
monocytogenes ATCC 15313, Peptococcus niger ATCC 27731, Peptostreptococcus
anaerobius ATCC 27337, Peptostreptococcus prevotii ATCC 9321, Staphylococcus
aureus ATCC 25923, Staphylococcus epidermidis ATCC 14990, Staphylococcus
haemolyticus ATCC 29970, Staphylococcus saprophyticus ATCC 15305,
Streptococcus agalactiae ATCC 27591, Streptococcus anginosus ATCC 33397,
Streptococcus bovis ATCC 33317, Streptococcus constellatus ATCC 27823,
Streptococcus dysgalactiae ATCC 43078, Streptococcus gordonii ATCC 10558,
Streptococcus mitis ATCC 33399, Streptococcus mutans ATCC 25175,
Streptococcus oralis ATCC 35037, Streptococcus parauberis ATCC 6631,
Streptococcus pneumoniae ATCC 6303, Streptococcus pyogenes ATCC 19615,
Streptococcus salivarius ATCC 7073, Streptococcus sanguinis ATCC 10556,
Streptococcus uberis ATCC 19436). These microbial species included 15 species
of
streptococci and many members of the normal vaginal and anal floras. In
addition, 40
GBS isolates of human origin, whose identification was confirmed by the Latex
agglutination test (Streptex, Murex), were also used to evaluate the ubiquity
of the
assay.
For determination of the sensitivities (i.e., the minimal number of genome
copies that could be detected) for conventional and LightCyclerTM PCR assays,
serial
10-fold or 2-fold dilutions of purified genomic DNA from 5 GBS ATCC strains
were
used.
Results
Evaluation of the GBS-specific conventional and LightCyclerTM PCR assay.
The specificity of the two assays demonstrated that only DNAs from GBS strains
could be amplified. Both PCR assays did not amplify DNAs from any other
bacterial
species tested including 14 streptococcal species other than GBS as well as
phylogenetically related species belonging to the genuses Enterococcus,
Peptostreptococcus and Lactococcus. Important members of the vaginal or anal
flora,
including coagulase-negative staphylococci, Lactobacillus sp., and Bacteriodes
sp.
were also negative with the GBS-specific PCR assay. The LightCyclerTM PCR
assays
detected only GBS DNA by producing an increased fluorescence signal which was
interpreted as a positive PCR result. Both PCR methods were able to amplify
all of
GBS clinical isolates, showing a perfect correlation with the phenotypic
identification methods.
40 The sensitivity of the assay was determined by using purified genomic DNA
CA 02307010 2000-OS-19
from the 5 ATCC strains of GBS. The detection limit for all of these 5 strains
was
one genome copy of GBS. The detection limit of the assay with the
LightCyclerTM
was 3.5 fg of genomic DNA (corresponding to 1-2 genome copies of GBS). These
results confirmed the high sensitivity of our GBS-specific PCR assay.
Direct Detection of GBS from anovaginal specimens. Among 66 anovaginal
specimens tested, 12 were positive for GBS by culture. 11 of them were also
identified by both PCR assays. The sensitivity of both PCR methods with
vaginal/anal specimens for identifying colonization status in pregnant women
at
delivery was 91.7°lo when compared to culture results. The specificity
and positive
predictive values were both 100% and the negative predictive value was 97.8%.
The
time for obtaining results was approximately 45 min for LightCyclerTM PCR,
approximately 100 min for conventional PCR and 48 hours for culture.
Conclusion
We have developed for the detection of GBS two PCR assays (conventional and
LightCyclerTM) which are specific (i.e., no amplification of DNA from a
variety of
bacterial species other than GBS) and sensitive (i.e., able to detect around 1
genome
copy for several reference ATCC strains of GBS). Both PCR assays are able to
detect
GBS directly from anovaginal specimens in a very short turnaround time. Using
the
real-time PCR assay on LightCyclerTM, we can detect GBS carriage in pregnant
women at delivery within 45 minutes.
EXAMPLE 15:
Simultaneous identification of Streptococcus pyo~enes and its pyro~enic
exotoxin A.
The rapid detection of Streptococcus pyogenes and of its pyrogenic exotoxin A
is of
clinical importance. We developed a multiplex assay which permits the
detection of
strains of S. pyogenes carrying the pyrogenic toxin A gene, which is
associated with
scarlet fever and other pathologies. In order to specifically detect S.
pyogenes,
nucleotide sequences of the pyrrolidone carboxylyl peptidase gene (pcp) were
aligned
to design PCR primers Spy291 (SEQ ID NO. 1211) and Spy473 (SEQ ID NO. 1210).
Next, we designed primers for the specific detection of the pyrogenic exotoxin
A.
Nucleotide sequences of the speA gene, carried on the bacteriophage T12, were
aligned as shown in Annex XXIII to design PCR primers Spytx814 (SEQ ID NO.
994) and Spytx 927 (SEQ ID NO. 995).
The primer pairs : oligos SEQ ID NOs. 1210-1211, yielding an amplification
product
of 207 bp, and oligos SEQ ID NOs. 994-995, yielding an amplification product
of
135 bp, were used in the PCR assay.
51
CA 02307010 2000-OS-19
PCR amplification was carned out using 0.4 ,uM of both pairs of primers, 2.5
mM
MgCl2, BSA 0.05 ,uM , dNTP 0.2 ~,M (Pharmacia), lOX PCR reaction buffer
(Promega), 0.025 U/ml Taq DNA polymerase (Promega) combined with TaqStart
(Clontech Laboratories Inc.), and 1 ,ul of genomic DNA sample in a final
volume of
20 ~,1. PCR amplification was performed using a PTC-200 thermal cycler (MJ
Research). The optimal cycling conditions for maximum sensitivity were 3
minutes at
94 °C for initial denaturation, then forty cycles of two steps
consisting of 1 second at
95 °C and 30 seconds at 63 °C, followed by a final step of 2
minutes at 72 °C.
l0 Detection of the PCR products was made by agarose gel (2%) electrophoresis
containing 0.25 ~,g/ml of ethidium bromide. Visualization of the PCR products
was
made under UV at 254 nm.
The detection limit was less than 5 genome copies for both S. pyogenes and its
pyrogenic exotoxin A. The assay was specific for pyrogenic exotoxin A-
producing S.
pyogenes : strains of the 27 other species of Streptococcus tested were all
negative, as
well as 20 strains of various gram-positive and gram-negative bacterial
species.
A similar approach was used to design an alternative set of speA-specific
primers
(SEQ ID NOs. 996 to 998, see Annex XXIV). In addition, another set of primers
based on the tuf gene (SEQ ID NOs. 999 to 1001, see Annex XXV) could be used
to
specifically detect Streptococcus pyogenes.
EXAMPLE 16
Real-time identification of Shiya toxin-producing bacteria Shiga toxin-
producing
Escherichia coli and Shigella dysenteriae cause bloody diarrhea. Currently,
identification relies mainly on the phenotypic identification of S.
dysenteriae and E.
coli serotype 0157:H7. However, other serotypes of E. coli are increasingly
found to
be producers of type 1 and/or type 2 Shiga toxins. Two pairs of PCR primers
targeting highly conserved regions present in each of the Shiga toxin genes
stxl and
stx2 were designed to amplify all variants of those genes (see Annexes XXVI
and
XXVII). The first primer pair, oligonucleotides 1 SLT224 (SEQ ID NO. 1081 )
and
1SLT385 (SEQ ID NO. 1080), yields an amplification product of 186 by from the
stxl gene. For this amplicon, the 1SLTB 1-Fam (SEQ ID NO. 1084) molecular
beacon
was designed for the specific detection of stx~ using the fluorescent label 6-
carboxy-
fluorescein. A second pair of PCR primers, oligonucleotides 2SLT537 (SEQ ID
NO.
1078) and 2SLT678b (SEQ ID NO. 1079), yields an amplification product of 160
by
from the stx2 gene. Molecular beacon 2SLTB 1-Tet (SEQ ID NO. 1085) was
designed
for the specific detection of stx2 using the fluorescent label 5-tetrachloro-
fluorescein.
52
CA 02307010 2000-OS-19
Both primer pairs were combined in a multiplex PCR assay.
PCR amplification was carried out using 0.8 ~.M of primers pair SEQ ID NOs.
1080-
1081, 0.5 ~,M of primer pair SEQ ID NOs. 1078-1079, 0.3 ~,M of each molecular
beacon, 8 mM MgCl2, 12.25 ~.g BSA, 0.2 mM dNTPs (Pharmacia), 50 mM Tris-HCI,
16 mM NH4S04, 1X TaqMaster (Eppendorf), 2.5 U KlenTaql DNA polymerise (AB
Peptides) combined with TaqStart (Clontech Laboratories Inc.), and 1 ~.1 of
genomic
DNA sample in a final volume of 25 ,ul. PCR amplification was performed using
a
SmartCycler thermal cycler (Cepheid). The optimal cycling conditions for
maximum
sensitivity were 60 seconds at 95 °C for initial denaturation, then 45
cycles of three
steps consisting of 10 seconds at 95 °C, 15 seconds at 56 °C and
5 seconds at 72 °C.
Detection of the PCR products was made in real-time by measuring the
fluorescent
signal emitted by the molecular beacon when it hybridizes to its target at the
end of
the annealing step at 56 °C.
The detection limit was the equivalent of less than 5 genome copies. The assay
was
specific for the detection of both toxins, as demonstrated by the perfect
correlation
between PCR results and the phenotypic characterization performed using
antibodies
specific for each Shiga toxin type. The assay was successfully performed on
several
Shiga toxin-producing strains isolated from various geographic areas of the
world,
including 10 0157:H7 E. coli, 5 non-0157:H7 E. coli and 4 S. dysenteriae.
EXAMPLE 17:
Development of a PCR assay for the detection and identification of
staphylococci at
genus and species levels and its associated mecA gene. The Staphylococcus
genus-
specific PCR primers described in Example 7 (SEQ ID NOs. 553 and 575) were
used
in multiplex with the mecA-specific PCR primers described in previous US
patent
serial no. 5,994,066 (SEQ ID NOs. 261 and 262 in the said patent) as well as
with the
S. aureus-specific PCR primers SEQ ID NOs. 152 and 153 described in the said
patent. Sequence alignment analysis of 10 publicly available mecA gene
sequences
were performed to design an internal probe for mecA (SEQ ID NO. 1177). An
internal probe was also designed for the S. aureus-specific amplicon (SEQ ID
NO
1234). PCR amplification and agarose gel analysis of the amplified products
were
performed as described in Example 7, with the exception that 0.4 ~.M (each) of
the
two Staphylococcus genus-specific primers (SEQ ID NOs. 553 and 575) and 0.4
~.M
(each) of the mecA gene primers and 0.4 ~.M (each) of the S. aureus-specific
primers
were used in the PCR mixture. The specificity of the multiplex assay with 40-
cycle
PCR protocols was verified by using purified genomic DNA from five methicillin-
resistant and fifteen methicillin-sensitive staphylococcal strains. The
sensitivity of the
53
CA 02307010 2000-OS-19
multiplex assay with 40-cycle PCR protocols was determined by using purified
genomic DNA from five methicillin-resistant and seven methicillin-sensitive
staphylococcal strains. A detection limit of 3-5 genome copies was obtained,
depending on the staphylococcal species tested. Furthermore, the mecA-specific
internal probe and the S. aureus-specific internal probe combined with (i) the
species-
specific internal probes, (ii) the genus-specific internal probes and (iii)
the coagulase-
negative staphylococci specific internal probes (described in Example 7) were
able to
recognize three methicillin-resistant staphylococcal strains with high
sensitivity and
specificity.
The format of the assay is not limited to the one described above. A person
skilled in
the art could adapt the assay for different formats such as PCR with real-time
detection using molecular beacon probes. Molecular beacon probes designed to
be
used in this assay include, but are not limited to, SEQ ID NO. 1232 for
detection of
the S. aureus-specific amplicon, SEQ ID NO. 1233 for detection of coagulase-
negative staphylococci and SEQ ID NO. 1231 for detection of mecA.
EXAMPLE 18:
Seduencing of pbpl a, pbp2b and pbp2x genes of Streptoccoccus pneumoniae. The
comparison of publicly available pbpl a, pbp2b and pbp2x sequences from a
variety
of S. pneumoniae strains revealed conserved regions allowing the design of PCR
primers able to amplify pbpl a, pbp2b, and pbp2x sequences of several strains
of S.
pneumoniae having various levels of resistance to penicillin and third-
generation
cephalosporins. Using primer pairs Spnpbp 1 a876 and Spnpbp 1 a2163 (SEQ ID
NOs.
1125 and 1126), Spnpbp2b580 and Spnpbp2b2045 (SEQ ID NOs. 1142 and 1143),
and Spnpbp2x469 and Spnpbp2x2212 (SEQ ID NOs. 1146 and 1147), it was possible
to amplify and sequence pbpla sequences SEQ ID NOs. 1004-1018, pbp2b
sequences SEQ ID NOs. 1019-1033, and pbp2x sequences SEQ ID NOs. 1034-1048.
Six other PCR primers (SEQ ID NOs. 1127-1128, 1144-1145, 1148-1149) were also
designed and used to complete the sequencing of pbpl a, pbp2b and pbp2x
amplification products.
EXAMPLE 19:
SeQuencin~ of hexA genes of Streptococcus species. The hexA sequence of S.
pneumoniae described in previous US patent serial no. 5,994,066 (SEQ ID NO. 31
in
the said patent, SEQ ID NO. 1183 in the present application) allowed the
design of a
PCR primer (SEQ ID NO. 1182) which was used with primer Spn1401 described in
previous US patent serial no. 5,994,066 (SEQ ID NO. 156 in the said patent,
SEQ ID
54
CA 02307010 2000-OS-19
NO. 1179 in the present application) to amplify the hexA gene of one strain of
S.
oralis, three strains of S. mitis and four strains of S. pneumoniae (Annex
XLII). Using
primers SEQ ID NO. 1179 and SEQ ID NO. 1182, it was possible to amplify and
sequence S. pneumoniae hexA (SEQ ID NOs. 1184-1187), S. mitis hexA (SEQ ID
NOs. 1189-1191) and S. oralis hexA (SEQ ID NO. 1188).
EXAMPLE 20:
Development of a multiplex PCR assay for the detection of Streptococcus
pneumoniae and its penicillin resistance genes.
Material and Methods
Bacterial strains. The specificity of the multiplex PCR assay was verified by
using a panel of ATCC (American Type Culture Collection) reference strains
consisting of 33 gram-negative and 67 gram-positive bacterial species (Table
13). In
addition, 55 clinical isolates of Streptococcus pneumoniae and 16 strains of
S. mitis
and 3 strains of S. oralis from the microbiology laboratory of the Centre
Hospitalier
Universitaire de Quebec, Pavillon Centre Hospitalier de 1'Universite Laval
(CHUL)
(Ste-Foy, Quebec, Canada) and from the Laboratoire de same publique du Quebec
(LSPQ) (Sainte-Anne de Bellevue, Quebec, Canada) were also tested to further
validate the Streptococcus pneumoniae-specific PCR assay. The penicillin MICs
(minimal inhibitory concentration ) of 55 isolates were measured by the broth
dilution method according to the recommended protocol of NCCLS.
PCR primers and internal probes. The comparison of hexA sequences from a
variety of streptococcal species described in Example 19 (SEQ ID NOs. 1184-
1191)
allowed the selection of a PCR primer specific for S. pneumoniae, Spnhexal613
(SEQ ID NO. 1181). This primer was used with the S. pneumoniae species-
specific
primer SEQ ID NO. 1179 to generate an amplification product of 213 by (Annex
XLII). The PCR primer SEQ ID NO. 1181 is located 87 nucleotides downstream on
the hexA sequence compared to the original S. pneumoniae species-specific PCR
primer Spn1515 described in previous US patent serial no. 5,994,066 (SEQ ID
NO.
157 in the said patent). These modifications were done to ensure the design of
the S.
pneumoniae species-specific internal probe according to the new hexA sequences
of
several close streptococcal species (SEQ ID NOs. 1184-1191). The comparison of
pbpl a sequences from S. pneumoniae strains with various levels of penicillin
resistance allowed the identification of amino acid substitutions Ile-459 to
Met and
CA 02307010 2000-OS-19
Ser-462 to Ala that occur in isolates with high-level penicillin resistance
(MICs >_
1~g/ml), and amino acid substitutions Ser-575 to Thr, Gln-576 to Gly and Phe-
577 to
Tyr that are common to all penicillin-resistant isolates with MICs >_ 0.25
~g/ml. As
shown in annex XXXI, PCR primer pair Spnpbp 1 a 1365 and Spnpbp 1 a 1747 (SEQ
ID
NOs. 1130 and 1131 ) were designed to detect high-level penicillin resistance
(MICs
>_ l~g/m1), whereas PCR primer pair Spnpbp1a1591 (SEQ ID NO. 1129) and SEQ ID
NO. 1131 were designed to detect intermediate-level penicillin resistance
(0.25 <_
MICs <_ O.S~.g/ml ).
The comparison of hexA sequences allowed the design of an internal probe
specific
for S. pneumoniae (SEQ ID NO. 1180) (Annex XLII). The range of mismatches
between the S. pneumoniae-specific 213-by amplicon was from 2 to 5, in the
middle
of the 19-by probe. Five internal probes containing all possible mutations
were
designed to detect the high-level penicillin resistance 383-by amplicon (SEQ
ID NOs.
1197, 1217-1220) and five internal probes containing all possible mutations
were
designed to detect the 156-by amplicon which includes both high-level and
intermediate penicillin resistance (SEQ ID NOs. 1094, 1192, 1193, 1214 and
1216).
Design and synthesis of primers and probes, and detection of the hybridization
were
performed as described in Example 7. Annex XXXI illustrates one of the
internal
probes for detection of the high-level penicillin resistance 383-by amplicon
(SEQ ID
NO. 1197) and one of the internal probes for detection of the intermediate
level
penicillin resistance 156-by amplicon (SEQ ID NO. 1193).
PCR amplification. For all bacterial species, amplification was performed from
purified genomic DNA. One ~.1 of 0.1 nanogram of genomic DNA was transferred
directly to a 19 ~,1 PCR mixture. Each PCR reaction contained 50 mM KCI; 10 mM
Tris-HCl (pH 9.0); 0.1% Triton X-100; 2.5 mM MgCl2; 0.1 ~,M (each) of the S.
pneumoniae species-specific primers SEQ ID NO. 1179 and SEQ ID NO. 1181, 0.1
,uM of the penicillin resistance primer SEQ ID NO. 1129, 0.7 ~.M of the other
penicillin resistance primer SEQ ID NO. 1130, and 0.6 ~,M of the high-level
penicillin resistance primer SEQ ID NO. 1131; 200 ,uM (each) of the four
deoxynucleoside triphosphates; 3.3 ~.g/~,l bovine serum albumin (BSA); and 0.5
U
Taq polymerase coupled with TaqStartTM Antibody.
For determination of the sensitivities of the PCR assays, two-fold dilutions
of
purified genomic DNA were used to determine the minimal number of genome
copies which can be detected.
Results
Amplifications with the multiplex PCR assay. The specificity of the assay was
56
CA 02307010 2000-OS-19
assessed by performing 40-cycle PCR amplifications with the panel of gram-
positive
(67 species from 12 genera) and gram-negative (33 species from 17 genera)
bacterial
species listed in Table 13. All bacterial species tested other than S.
pneumoniae were
negative except S. mitis and S. oralis. Ubiquity tests were performed using a
collection of 55 clinical S. pneumoniae isolates provided by the microbiology
laboratory of the CHUL and the LSPQ, including high-level penicillin
resistance
(n=37), intermediate resistance (n=11 ) and sensitive (n=7) strains. There was
a
perfect correlation between PCR and standard susceptibility testing for 37
isolates
with high-level penicillin resistance and 7 penicillin-sensitive isolates.
Among 11
l0 isolates with intermediate penicillin resistance, 9 had intermediate
resistance based on
PCR but two isolates with MIC of 0.5 ~g/ml showed a high-level penicillin
resistance
based on genotyping. This demonstrated that MIC of 0.5 ~,g/ml may represent
intermediate or high-level penicillin resistance. In general, there was a good
correlation between the genotype and classical identification schemes.
The sensitivity of the S. pneumoniae-specific assay with 40-cycle PCR
protocols
was determined by using purified genomic DNA from 9 isolates of S. pneumoniae.
The detection limit was 2-10 genome copies, depending on the pbp amplification
pattern.
Hybridization between multiplex PCR amplicons and internal probes. The S.
pneumoniae-specific internal probe did not hybridize to the S. mitis and S.
oralis
non-specific PCR amplification products generated from the S. pneunoniae-
specific
PCR primers. More precisely, this specificity was tested by using genomic DNA
purified from 16 strains of S. mitis and from 3 strains of S. oralis. The
ubiquity of the
assay was tested by using genomic DNA from 15 strains of S. pneumoniae. In
summary, the combination of the PCR and hybridization assays results in a
highly
specific test for the detection of penicillin-resistant Streptococcus
pneumoniae.
3o EXAMPLE 21:
Sequencing of the vancomycin resistance vanA, vanCl , vanC2 and vanC3 ~. The
publicly available sequences of the vanes-vanA-vanX-vanY locus of transposon
Tn 1546 from E. faecalis, vanCl sequence from one strain of E. gallinarum,
vanC2
and vanC3 sequences from a variety of E. casseliflavus and Enterococcus
flavescens
strains, respectively, allowed the design of PCR primers able to amplify the
vanA,
vanCl, vanC2 and vanC3 sequences of several Enterococcus species. Using primer
pairs van6877 and van9106 (SEQ ID NOs. 1150 and 1155), vanCl-122 and vanCl-
1315 (SEQ ID NOs. 1110 and 1109), and vanC2C3-1 and vanC2C3-1064 (SEQ ID
NOs. 1108 and 1107), it was possible to amplify and sequence vanA sequences
57
CA 02307010 2000-OS-19
SEQ ID NOs. 1049-1057, vanCl sequences SEQ ID NOs. 1058-1059, vanC2
sequences SEQ ID NOs. 1060-1063 and vanC3 sequences SEQ ID NOs. 1064-1066,
respectively. Three other PCR primers (SEQ ID NOs. 1151-1154) were also
designed
and used to complete the sequencing of vanA amplification products.
EXAMPLE 22:
Development of a PCR assay for the detection and identification of enterococci
at
genus and species levels and its associated resistance genes vanA and vanB.
The
comparison of vanA and vanB sequences revealed conserved regions allowing the
design of PCR primers specific for both vanA and vanB sequences (Annex
XXXVIII). The PCR primer pair vanAB459 and vanAB830R (SEQ ID NOs. 1112
and 1111 ) was used in multiplex with the Enterococcus genus-specific primers
Encg313dF and Encg599c (SEQ ID NOs. 1137 and 1136) described in Example 11.
Sequence alignment analysis of vanA and van8 sequences revealed regions
suitable
for the design of internal probes specific for vanA (SEQ ID NO. 1170) and vanB
genes (SEQ ID NO. 1171 ). PCR amplification and agarose gel analysis of the
amplified products were performed as described in Example 11. The specificity
of the
multiplex assay with 40-cycle PCR was verified by using 0.1 nanogram of
purified
genomic DNA from a panel of bacteria listed in Table 10. The sensitivity of
the
multiplex assay with 40-cycle PCR was verified with three strains of E.
casseliflavus,
eight strains of E. gallinarum, two strains of E. flavescens, two vancomycin-
resistant
strains of E. faecalis and one vancomycin-sensitive strain of E. faecalis,
three
vancomycin-resistant strains of E. faecium and one vancomycin-sensitive strain
of E.
faecium, and one strain of each of the other enterococcal species listed in
Table 10. A
detection limit of 1-10 genome copies was obtained, depending on the
enterococcal
species tested. In addition to the species-specific internal probes described
in
Example 11, the vanA- and vanB-specific internal probes were able to recognize
vancomycin-resistant enterococcal species with high sensitivity, specificity
and
ubiquity and with a perfect correlation between the genotypic and phenotypic
analysis.
The format of the assay is not limited to the one described above. A person
skilled in
the art could adapt the assay for different formats such as PCR with real-time
detection using molecular beacon probes. Molecular beacon probes designed to
be
used in this assay include, but are not limited to, SEQ ID NO. 1236 for the
detection
of E. faecalis, SEQ ID NO. 1235 for the detection of E. faecium, SEQ ID NO.
1240
for the detection of vanA, and SEQ ID NO. 1241 for the detection of vanB.
EXAMPLE 23:
58
CA 02307010 2000-OS-19
Development of a multiplex PCR assay for detection and identification of
vancomycin-resistant Enterococcus faecalis, Enterococcus faecium, Enterococcus
gallinarum, Enterococcus cassel~avus, and Enterococcus ~lavescens. The
comparison of vanA and vanB sequences revealed conserved regions allowing
design
of a PCR primer pair (SEQ ID NOs. 1089 and 1090) specific for vanA sequences
(Annex XXVIII) and a PCR primer pair (SEQ ID NOs. 1095 and 1096) specific for
vanB sequences (Annex XXIX). The vanA-specific PCR primer pair (SEQ ID NOs.
1089 and 1090) was used in multiplex with the vanB-specific PCR primer pair
(SEQ
ID NOs. 1095 and 1096). The comparison of vanCl, vanC2 and vanC3 sequences
revealed conserved regions allowing design of PCR primers (SEQ ID NOs. 1101
and
1102) able to generate a 158-by amplicon specific for E. gallinarum, E.
casseliflavus
and E. flavescens (Annex XXX). The vanC-specific PCR primer pair (SEQ ID NOs.
1101 and 1102) was used in multiplex with the E. faecalis species-specific PCR
primer pair described in previous US patent serial no. 5,994,066 (SEQ ID NOs.
40
and 41 in the said patent) and with the E. faecium species-specific PCR primer
pair
described in previous patent application W098/20157 (SEQ ID NOs. 1 and 2 in
the
said application). For both multiplexes, the optimal cycling conditions for
maximum
sensitivity and specificity were found to be 3 min. at 94 °C, followed
by forty cycles
of two steps consisting of 1 second at 95 °C and 30 seconds at 58
°C, plus a terminal
extension at 72 °C for 2 minutes. Amplification was monitored on
agarose gel
electrophoresis by staining the amplified DNA with ethidium bromide. The vanA-
specific PCR primer pair (SEQ ID NOs. 1089 and 1090), the vanB-specific
primers
pair (SEQ ID NOs. 1095 and 1096) and the vanC-specific primer pair (SEQ ID
NOs.
1101 and 1102) were tested for their specificity by using 0.1 nanogram of
purified
genomic DNA from a panel of 5 vancomycin-sensitive Enterococcus species, 3
vancomycin-resistant Enterococcus species, 13 other gram-positive bacteria and
one
gram-negative bacterium. Specificity tests using the E. faecium species-
specific PCR
primer pair described in previous patent application W098/20157 (SEQ ID NOs. 1
and 2 in the said application) and the E. faecalis species-specific PCR primer
pair
described in previous US patent serial no. 5,994,066 (SEQ ID NOs. 40 and 41 in
the
said patent) were performed on a panel of 37 gram-positive bacterial species.
All
Enterococcus strains were amplified with high specificity and there was a
perfect
correlation between the genotypic and phenotypic analysis. Finally, the
sensitivity of
the assays was determined for several strains of E. gallinarum, E.
casseliflavus, E.
flavescens and vancomycin-resistant E. faecalis and E. faecium. Using each of
the E.
faecalis and E. faecium species-specific PCR primer pairs as well as vanA,
vanB and
vanC-specific PCR primers used alone or in multiplex as described above, the
sensitivity ranged from 1 to 10 genome copies.
The format of the assay is not limited to the one described above. A person
skilled in
the art could adapt the assay for different formats such as PCR with real-time
59
CA 02307010 2000-OS-19
detection using molecular beacon probes. Molecular beacon probes designed to
be
used in this assay include, but are not limited to, SEQ ID NO. 1238 for the
detection
of E. faecalis, SEQ ID NO. 1237 for the detection of E. faecium, SEQ ID NO.
1239
for the detection of vanA, and SEQ ID NO. 1241 for the detection of vanB.
EXAMPLE 24:
Universal amplification involving the EF-G (fusA) subdivision of tuf
sequences. As
shown in Figure 3, primers SEQ ID NOs. 1228 and 1229 were designed to amplify
the region between the end of fusA and the beginning of tuf genes in the str
operon.
Genomic DNAs from a panel of 35 strains were tested for PCR amplification with
those primers. The following strains showed a positive result: Abiotrophia
adiacens
ATCC 49175, Abiotrophia defectiva ATCC 49176, Bacillus subtilis ATCC 27370,
Closridium difficile ATCC 9689, Enterococcus avium ATCC 14025, Enterococcus
casseliflavus ATCC 25788, Enterococcus cecorum ATCC 43198, Enterococcus
faecalis ATCC 29212, Enterococcus faecium ATCC 19434, Enterococcus flavescens
ATCC 49996, Enterococcus gallinarum ATCC 49573, Enterococcus solitarius
ATCC 49428, Escherchia coli ATCC 11775, Haemophilus influenzae ATCC 9006,
Lactobacillus acidophilus ATCC 4356, Peptococcus niger ATCC 27731, Proteus
mirabilis ATCC 25933, Staphylococcus aureus ATCC 43300, Staphylococcus
auricularis ATCC 33753, Staphylococcus capitis ATCC 27840, Staphylococcus
epidemidis ATCC 14990, Staphylococcus haemolyticus ATCC 29970,
Staphylococcus hominis ATCC 27844, Staphylococcus lugdunensis ATCC 43809,
Staphylococcus saprophyticus ATCC 15305, Staphylococcus simulans ATCC 27848,
and Staphylococcus warneri ATCC 27836. This primer pair could amplify
additional
bacterial species; however, there was no amplification for some species,
suggesting
that the PCR cycling conditions could be optimized or the primers modified.
For
example, SEQ ID NO. 1227 was designed to amplify a broader range of species.
In addition to other possible primer combinations to amplify the region
covering fusA
and tuf, Figure 3 illustrates the positions of amplification primers SEQ ID
NOs. 1221
1227 which could be used for universal amplification of fusA segments. All of
the
above mentioned primers (SEQ ID NOs. 1221-1229) could be useful for the
universal
and/or the specific detection of bacteria.
EXAMPLE 25:
DNA fragment isolation from Staphylococcus saprophyticus by arbitraril~primed
PCR. DNA sequences of unknown coding potential for the species-specific
detection
and identification of Staphylococcus saprophyticus were obtained by the method
of
arbitrarily primed PCR (AP-PCR).
AP-PCR is a method which can be used to generate specific DNA probes for
microorganisms (Fani et al., 1993, Molecular Ecology 2:243-250). A description
of
the AP-PCR protocol used to isolate a species-specific genomic DNA fragment
from
Staphylococcus saprophyticus follows. Twenty different oligonucleotide primers
of
CA 02307010 2000-OS-19
nucleotides in length (all included in the AP-PCR kit OPAD (Operon
Technologies, Inc., Alameda, CA)) were tested systematically with DNAs from 5
bacterial strains of Staphylococcus saprophyticus as well as with bacterial
strains of
27 other staphylococcal (non-S. saprophyticus) species. For all bacterial
species,
5 amplification was performed directly from one ~,L (0.1 ng/~.L) of purified
genomic
DNA. The 25 ~,L PCR reaction mixture contained 50 mM KCI, 10 mM Tris-HCl (pH
9.0), 0.1 % Triton X-100, 2.5 mM MgCl.2, 1.2 ~,M of only one of the 20
different AP-
PCR primers OPAD, 200 ~,M of each of the four dNTPs, 0.5 U of Taq DNA
polymerase (Promega Corp., Madison, Wis.) combined with TaqStartTM antibody
10 (Clontech Laboratories Inc., Palo Alto, CA). PCR reactions were subjected
to cycling
using a MJ Research PTC-200 thermal cycler as follows: 3 min at 96 °C
followed by
42 cycles of 1 min at 94 °C for the denaturation step, 1 min at 31
°C for the annealing
step and 2 min at 72 °C for the extension step. A final extension step
of 7 min at 72
°C followed the 42 cycles to ensure complete extension of PCR products.
Subsequently, twenty microliters of the PCR-amplified mixture were resolved by
electrophoresis on a 1.5 % agarose gel containing 0.5 ~,g/mL of ethidium
bromide.
The size of the amplification products was estimated by comparison with a 50-
by
molecular weight ladder.
Amplification patterns specific for Staphylococcus saprophyticus were observed
with
the AP-PCR primer OPAD-16 (sequence: 5'-AACGGGCGTC-3'). Amplification with
this primer consistently showed a band corresponding to a DNA fragment of
approximately 380 by for all Staphylococcus saprophyticus strains tested but
not for
any of the other staphylococcal species tested.
The band corresponding to the 380 by amplicon, specific and ubiquitous for S.
saprophyticus based on AP-PCR, was excised from the agarose gel and purified
using
the QIAquick~ gel extraction kit (QIAGEN Inc.). The gel-purified DNA fragment
was cloned into the T/A cloning site of the pCR 2.1TM plasmid vector
(Invitrogen
Inc.) using T4 DNA ligase (New England BioLabs). Recombinant plasmids were
transformed into E. coli DHSa competent cells using standard procedures. All
reactions were performed according to the manufacturer's instructions. Plasmid
DNA
isolation was done by the method of Birnboim and Doly (Nucleic Acid Res.,
1979,
7:1513-1523) for small-scale preparations. All plasmid DNA preparations were
digested with the EcoRI restriction endonuclease to ensure the presence of the
approximately 380 by AP-PCR insert into the plasmid. Subsequently, a large-
scale
and highly purified plasmid DNA preparation was performed from two selected
clones shown to carry the AP-PCR insert by using the QIAGEN plasmid
purification
kit (midi format). These large-scale plasmid preparations were used for
automated
DNA sequencing.
The 380 by nucleotide sequence was determined for three strains of S.
saprophyticus
(SEQ ID NOs. 74, 1093, and 1198). Both strands of the AP-PCR insert from the
two
selected clones were sequenced by the dideoxynucleotide chain termination
sequencing method with SP6 and T7 sequencing primers by using the Applied
Biosystems automated DNA sequencer (model 373A) with their PRISMTM
SequenaseRTM Terminator Double-stranded DNA Sequencing Kit (Perkin-Elmer
Corp., Applied Biosystems Division, Foster City, CA).
61
CA 02307010 2000-OS-19
Optimal species-specific amplification primers (SEQ ID NOs. 1208 and 1209)
have
been selected from the sequenced AP-PCR Staphylococcus saprophyticus DNA
fragments with the help of the primer analysis software OligoTM 4.0 (National
BioSciences Inc.). The selected primers were tested in PCR assays to verify
their
specificity and ubiquity. Data obtained with DNA preparations from reference
ATCC
strains of 49 gram-positive and 31 gram-negative bacterial species, including
28
different staphylococcal species, indicate that the selected primer pairs are
specific
for Staphylococcus saprophyticus since no amplification signal has been
observed
with DNAs from the other staphylococcal or bacterial species tested.
l0
This invention has been described herein above, and it is readily apparent
that
modifications can be made thereto without departing from the spirit of this
invention.
These modifications are under the scope of this invention, as defined in the
appended
claims.
62
CA 02307010 2000-OS-19
Table 1. Distribution (%) of nosocomial pathogens for various human infections
in USA
(1990-1992) .
Pathogen UTI2 SS13 BS14 Pneumonia CSFS
Escherichia coli 27 9 5 4 2
Staphylococcus aureus 2 21 17 21 2
Staphylococcus epidermidis2 6 20 0 1
Enterococcus faecalis 16 12 9 2 0
Enterococcus faecium 1 1 0 0 0
Pseudomonas aeruginosa 12 9 3 18 0
Klebsiella pneumoniae 7 3 4 9 0
Proteus mirabilis 5 3 1 2 0
Streptococcus pneumoniae0 0 3 1 18
Group B Streptococci 1 1 2 1 6
Other streptococci 3 5 2 1 3
Haemophilus influenzae 0 0 0 6 45
Neisseria meningitidis 0 0 0 0 14
Listeria monocytogenes 0 0 0 0 3
Other enterococci 1 1 0 0 0
Other staphylococci 2 8 13 2 0
Candida albicans 9 3 5 5 0
Other Candida 2 1 3 1 0
Enterobacter sp. 5 7 4 12 2
Acinetobacter sp. 1 1 2 4 2
Citrobacter s p. 2 1 1 1 0
Serratia marcescens 1 1 1 3 1
Other Klebsiella 1 1 1 2 1
Others 0 6 4 5 0
Data recorded by the National Nosocomial Infections Surveillance (NNIS) from
80 hospitals (Emori and
Gaynes, 1993, Clin. MicrobioL Rev., 6:428-442).
2
Urinary tract infection.
3
Surgical site infection.
4
Bloodstream infection.
s
Cerebrospinal fluid.
63
CA 02307010 2000-OS-19
Table 2. Distribution (%) of bloodstream infection pathogens in Quebec (1995),
Canada (1992),
UK (1969-1988) and USA (1990-1992).
Organism Quebec Canada2 UK3 USA4
Community-Hospital-Hospital-
acquired acquiredacquired
E, coii 15.6 53.8 24.8 20.3 5.0
S. epidermidis and
5
other CONS 25.8 - 0.5 7.2 31.0
S. aureus 9.6 - 9.7 19.4 16.0
S. pneumoniae 6.3 - 22.5 2.2 -
E. faecalis 3.0 - 1.0 4.2
E, faecium 2.6 - 0.2 0.5 -
Enterococcus sp. - - - - g,0
H. influenzae 1.5 - 3.4 0.4 -
P. aeruginosa 1.5 8.2 1.0 8.2 3.0
K. pneumoniae 3.0 11.2 3.0 9.2 4.0
P. mirabilis - 3.9 2.8 5.3 1.0
S. pyogenes - - 1.9 0.9 -
Enterobacter sp. 4.1 5.5 0.5 2.3 4.0
Candida sp. 8.5 - - 1.0 8.0
Others 18.5 17.4 28.7 18.9 19.0
Data obtained for 270 isolates collected fUniversiteLaval (CHUL)
at the Centre Hospitalier de during a 5
month period (May to October 1995).
z
Data from 10 hospitals throughout ng 941
Canada representi gram-negative
isolates.
(Chamberland
et al.,
1992, Clin. Infect. Dis., 15:615-628).
3 Data from a 20-year study (1969-1988)4000 isolates.(Eykyn
for nearly et al.,
1990,
J. Antimicrob.
Chemother., Suppl. C, 25:41-58).
4
Data recorded by the National Nosocomial 80 hospitals
Infections Surveillance (NNIS) from (Emori and
Gaynes, 1993, Clin. Microbiol. Rev.,
6:428-442).
s
Coagulase-negative staphylococci.
64
CA 02307010 2000-OS-19
Table 3. Distribution of positive and negative clinical specimens tested at
the microbiology laboratory
of the CHUL (February 1994 - January 1995).
Clinical specimens No. of samples % of positive % of negative
and/or sites tested (%) specimens specimens
Urine 17,981 (54.5) 19.4 80.6
Blood culture/marrow 10,010 (30.4) 6.9 93.1
Sputum 1,266 (3.8) 68.4 31.6
Superficial pus 1,136 (3.5) 72.3 27.7
Cerebrospinal fluid 553 (1.7) 1.0 99.0
Synovial fluid 523 (1.6) 2.7 97.3
Respiratory tract 502 (1.5) 56.6 43.4
Deep pus 473 (1.4) 56.8 43.2
Ears 289 (0.9) ~ 47.1 52.9
Pleural and pericardial 132 (0.4) 1.0 99.0
fluid
Peritoneal fluid 101 (0.3) 28.6 71.4
Total: 32,966 (100.0) 20.0 80.0
CA 02307010 2000-OS-19
Table 4. Non-limitating example of microbial species for which tuf and/or atpD
and/or recA sequences
are used in the present invention.
Bacterial species
Achromobacterxylosoxidans subsp. Chlamydia trachomatis
denitrificans
Acetobacterium woodi Chlorobium vibrioforme
Acetobacter aceti Chloroflexus aurantiacus
Acetobacter altoacetigenes 65 Chryseobacterium meningosepticum
Acetobacter polyoxogenes Citrobacter amalonaticus
Acholeplasma laidlawii Citrobacter braakii
Acidiphilum facilis Citrobacter farmeri
Acinetobacter baumannii Citrobacter freundii
Acinetobacter calcoaceticus 70 Citrobacter koseri
Acinetobacter Iwotfii Citrobacter sedlakii
Actinomyces meyeri Citrobacter werkmanii
Aerococcus viridans Citrobacter youngae
Aeromonas salmonicida Clostridium acetobutylicum
Agrobacterium tumefaciens 75 Clostridium beijerinckii
Alcaligenes faecalis Clostridium bifermentans
Allochromatium vinosum Clostridium botulinum
Anabaena variabilis Clostridium difficile
Anacystis nidulans Clostridium innocuum
Anaerorhabdus furcosus 80 Clostridium histolyticum
Aquifex aeolicus Clostridium novyi
Aquifex pyrophilus Clostridium septicum
Azotobacter vinelandii Clostridium perfringens
Bacillus anthracis Clostridium ramosum
Bacillus cereus 85 Clostridium sordellii
Bacillus firmus Clostridium tertium
Bacillus halodurans Clostridium tetani
Bacillus megaterium Comamonas acidovorans
Bacillus stearothermophilus Corynebacterium bovis
Bacillus subtilis 90 Corynebacterium cervicis
Bacteroides distasonis Corynebacterium diphtheriae
Bacteroides fragilis Corynebacterium flavescens
Bacteroides ovatus Corynebacterium glutamicum
Bacteroides vulgatus Corynebacterium kutscheri
Bartonella henselae 95 Corynebacterium minutissimum
Bifidobacterium adolescentis Corynebacterium mycetoides
Bifidobacterium breve Corynebacterium pseudodiphtheriticum
Bifidobacterium dentium Corynebacterium pseudogenitalium
Bifidobacterium longum Corynebacterium pseudotuberculosis
Blastochloris viridis 100 Corynebacterium renale
Borrelia burgdon'eri Corynebacterium ulcerans
Bordetella pertussis Corynebacterium urealyticum
Bordetella bronchiseptica Corynebacterium xerosis
Branhamella catarrhalis Coxiella burnetii
Brucella abortus 105 Cytophaga lytica
Brevibacterium linens Deinococcus radiodurans
Brevibacterium flavum Deinonema sp.
Buchnera aphidicola Edwardsiella hoshinae
Burkholderia cepacia Edwardsiella tarda
Burkholderia mallei 110 Ehrlichia canis
Burkholderia pseudomallei Ehrlichia risticii
Campylobacter jejuni Eikenella corrodens
Cedecea davisae Enterobacter aerogenes
Cedecea lapagei Enterobacter agglomerans
Cedecea neteri 115 Enterobacter amnigenus
Chlamydia pneumoniae Enterobacter asburiae
Chlamydia psittaci Enterobacter cancerogenus
66
CA 02307010 2000-OS-19
Table 4. Non-limitating example of microbial species for which tuf and/or atpD
and/or recA sequences
are used in the present invention (continued).
Bacterial species (continued)
Enterobacter cloacae Helicobacter pylori
Enterobacter gergoviae 65 Herpetoshiphon aurantiacus
Enterobacter hormaechei Kingella kingae
Enterobacter sakazakii Klebsiella ornithinolytica
Enterococcus avium Klebsiella oxyfoca
Enterococcus casseliflavus Klebsiella planticola
Enterococcus cecorum 70 Klebsiella pneumoniae subsp.
ozaenae
Enterococcus columbae Klebsiella pneumoniae subsp.
pneumoniae
Enterococcus dispar Klebsiella pneumoniae subsp.
Enterococcus durans rhinoscleromatis
Enterococcus faecalis Kluyvera ascorbata
Enterococcus faecium 75 Kluyvera cryocrescens
Enterococcus flavescens Kluyvera georgiana
Enterococcus gallinarum Lactobacillus acidophilus
Enterococcus hirae Lactobacillus garvieae
Enterococcus malodoratus Lactobacillus paracasei
Enterococcus mundtii 80 Lactobacillus casei subsp.
casei
Enterococcus pseudoavium Lactococcus lactis subsp. lactis
Enterococcus raffinosus Leclercia adecarboxylata
Enterococcus saccharolyticus Legionella micdadei
Enterococcus solitarius Legionella pneumophila subsp.
pneumophila
Enterococcus sulfureus 85 Leminorella grimontii
Erwinia carotovora Leminorella richardii
Escherichia coli Leptospira biflexa
Escherichia fergusonii Leptospira interrogans
Escherichia hermannii Listeria monocytogenes
Escherichia vulneris 90 Macrococcus caseolyticus
Eubacterium lentum Magnetospirillum magnetotacticum
Eubacterium nodatum Megamonas hypermegale
Ewingella americana Methanobacterium thermoautotrophicum
Francisella tularensis Methanococcus jannaschii
Frankia alni 95 Methanococcus vannielii
Fervidobacterium islandicum Methanosarcina barkeri
Fibrobacter succinogenes Methanosarcina jannaschii
Flavobacterium ferrigeneum Methylobacillus flagellatum
Flexistipes sinusarabici Methylomonas clara
Fusobacterium gonidiaformans 100 Micrococcus luteus
Fusobacterium necrophorum subsp. Micrococcus lylae
necrophorum
Fusobacterium nucleatum subsp. Mitsuokella multacida
polymorphum
Gardnerella vaginalis Mobiluncus curtisii subsp.
holmesii
Gemella haemolysans Moellerella fhermoacetica
Gemella morbillorum 105 Moellerella wisconsensis
Gloeobacter violaceus Moraxella osloensis
Gloeothece sp. Morganella morganii subsp.
morganii
Gluconobacter oxydans Mycobacterium avium
Haemophilus actinomycetemcomitans Mycobacterium bovis
Haemophilus aphrophilus 110 Mycobacterium gordonae
Haemophilus ducreyi Mycobacterium leprae
Haemophilus haemolyticus Mycobacterium tuberculosis
Haemophilus influenzae Mycoplasma capricolum
Haemophilus parahaemolyticus Mycoplasma gallisepticum
Haemophilus parainfluenzae 115 Mycoplasma genitalium
Haemophilus paraphrophilus Mycoplasma hominis
Haemophilus segnis Mycoplasma pirum
Hafnia alvei Mycoplasma mycetoides
Haloarcula marismortui Mycoplasma pneumoniae
Halobacterium salinarum 120 Mycoplasma pulmonis
Haloferax volcanii
67
CA 02307010 2000-OS-19
Table 4. Non-limitating example of microbial species for which tuf and/or atpD
and/or recA
sequences are used in the present invention (continued).
Bacterial species (continued)
Mycoplasma salivarium Rhodospirillum rubrum
Myxococcus xanthus 65 Ruminococcus albus
Neisseria animalis Salmonella bongori
Neisseria canes Salmonella choleraesuis subsp.
arizonae
Neisseria cinerea Salmonella choleraesuis subsp
choleraesuis
Neisseria cuniculi Salmonella choleraesuis subsp.
diarizonae
Neisseria elongata subsp. 70 Salmonella choleraesuis subsp.
elongata houtenae
Neisseria elongata subsp. Salmonella choleraesuis subsp.
intermedia indica
Neisseria flava Salmonella choleraesuis subsp.
salamae
Neisseria flavescens Serpulina hyodysenteriae
Neisseria gonorrhoeae Serratia ficaria
Neisseria lactamica 75 Serratia fonticola
Neisseria meningitides Serratia grimesii
Neisseria mucosa Serratia liquefaciens
Neisseria perflava Serratia marcescens
Neisseria pharynges Serratia odorifera
Neisseria polysaccharea 80 Serratia plymuthica
Neisseria sicca Serratia rubidaea
Neisseria subflava Shewanella putida
Neisseria weaveri Shewanella putrefaciens
Ochrobactrum anthropi Shigella boydii
Pantoea agglomerans 85 Shigella dysenteriae
Pantoea dispersa Shigella flexneri
Paracoccus denitrificans Shigella sonnei
Pasteurella multocida Spirochaeta aurantia
Pectinatus frisingensis Staphylococcus aureus
Peptococcus niger 90 Staphylococcus aureus subsp.
aureus
Peptostreptococcus anaerobius Staphylococcus auricularis
Peptostreptococcus asaccharolyticus Staphylococcus capitis subsp.
capitis
Peptostreptococcus prevotii Staphylococcus cohnii
Phormidium ectocarpi Staphylococcus epidermidis
Pirellula marina 95 Staphylococcus haemolyticus
Planobispora rosea Staphylococcus hominis
Plectonema boryanum Staphylococcus lugdunensis
Porphyromonas asaccharolytica Staphylococcus saprophyticus
Porphyromonas gingivalis Staphylococcus sciuri subsp.
sciuri
Pragia fontium 100 Staphylococcus simulans
Prevotella melaninogenica Staphylococcus warneri
Prevotella oralis Stigmatella aurantiaca
Prevotella ruminocola Stenotrophomonas maltophilia
Prochlorothrix hollandica Streptococcus acidominimus
Propionibacterium acnes 105 Streptococcus agalactiae
Propionigenium modestum Streptococcus anginosus
Proteus mirabilis Streptococcus bovis
Proteus penneri Streptococcus cricetus
Proteus vulgaris Streptococcus cristatus
Providencia alcalifaciens 110 Streptococcus downei
Providencia rettgeri Streptococcus dysgalactiae
Providencia rustigianii Streptococcus equi subsp.
equi
Providencia stuartii Streptococcus ferns
Pseudomonas aeruginosa Streptococcus gordonii
Pseudomonas fluorescens 115 Streptococcus macacae
Pseudomonas stutzeri Streptococcus mites
Psychrobacter phenylpyruvicus Streptococcus mutans
Rahnella aquatilis Streptococcus oralis
Rickettsia prowazekii Streptococcus parasanguinis
Rhodobacter capsulatus 120
Rhodobacter sphaerolides
68
CA 02307010 2000-OS-19
Table 4. Non-limitating example of microbial species for which tut and/or atpD
and/or recA
sequences are used in the present invention (continued).
Bacterial species (continued)
Streptococcus pneumoniae Aspergillus oryzae
Aspergillus flavus
Streptococcus pyogenes 65 Aspergillus fumigatus
Streptococcus ratti Aspergillus niger
Streptococcus salivarius Aureobasidium pullulans
Streptococcus salivarius subsp.
thermophilus
Streptococcus sanguinis Bipolaris hawaiiensis
Blastoschizomyces
capitatus
Streptococcus sobrinus 70 Candida albicans
Streptococcus suis Candida catenulata
Streptococcus uberis Candida dubliniensis
Streptococcus vestibularis Candida famata
Streptomyces anbofaciens Candida glabrata
Streptomyces aureofaciens 75 Candida guilliermondii
Streptomyces cinnamoneus Candida haemulonii
Streptomyces coelicolor Candida inconspicua
Streptomyces collinus Candida kefyr
Streptomyces lividans Candida krusei
Streptomyces ramocissimus 80 Candida lambica
Streptomyces rimosus Candida lusitaniae
Streptomyces venezuelae
Synechococcus sp. Candida norvegensis
Synechocystis sp. Candida parapsilosis
Tatumella ptyseos Candida rugosa
Taxeobacter occealus 85 Candida sphaerica
Thermoplasma acidophilum Candida tropicalis
Thermotoga maritima Candida utilis
Thermus apuaticus Candida viswanathii
Thermus thermophilus Candida zeylanoides
Thiobacillus ferrooxydans 90 Cladophialophora
carrionii
Thiomonas cuprina Coccidioides immitis
Trabulsiella guamensis Coprinus cinereus
Treponema pallidum Cryptococcus albidus
Ureaplasma urealyticum Cryptococcus humicolus
Veillonella parvula 95 Cryptococcus neoformans
Vibrio alginolyticus Cunninghamella bertholletiae
Vibrio anguillarum Curvularia lunata
Vibrio cholerae Emericella nidulans
IiVolinella succinogenes Exophiala jeanselmei
Xanthomonas citri 100 Eremothecium gossypii
Xanthomonas oryzae Fonsecaea pedrosoi
Xenorhabdus bovieni Fusarium oxysporum
Xenorhabdus nematophilus Geotrichum sp.
Yersinia bercovieri Histoplasma capsulatum
Yersinia enterocolitica 105 lssatchenkia orientalis
Yersinia frederikensii Kluyveromyces lactis
Yersinia intermedia Malassezia furfur
Yersinia pestis Malassezia pachydermatis
Yersinia pseudotuberculosis Malbranchea filamentosa
Yersinia rohdei 110 Metschnikowia pulcherrima
Yokenella regensburgei Microsporum audouinii
Zoogloea ramigera Mucor circinelloides
Neurospora crassa
Fungal species Paecilomyces lilacinus
115 Paracoccidioides
brasiliensis
Penicillium marneffei
Absidia corymbifera Phialaphora verrucosa
Absidia glauca Pichia anomala
Alternaria alternata
Arxula adeninivorans
69
CA 02307010 2000-OS-19
Table 4. Non-limitating example of microbial species for which tufand/or atpD
and/or recA
sequences are used in the present invention (continued).
Fungal species (continued)
Piedraia hortai Trypanosoma brucei subsp. brucei
Podospora anserina Trypanosoma congolense
Puccinia graminis 65 Trypanosoma cruzi
Pseudallescheria boydii
Rhizomucor racemosus
Rhizopus oryzae
Rhodotorula minuta
Rhodotorula mucilaginosa
Saccharomyces cerevisiae
Saksenaea vasiformis
Schizosaccharomyces pombe
Scopulariopsis koningii
Sporobolomyces salmonicolor
Sporothrix schenckii
Stephanoascus ciferrii
Syncephalastrum racemosum
Trichoderma reesei
Trichophyton mentagrophytes
Trichophyton tonsurans
Trichosporon cutaneum
Ustilago maydis
lNangiella dermatitidis
Yarrowia lipolytica
Parasitical species
Babesia bigemina
Babesia bovis
Babesia microti
Blastocystis hominis
Crithidia fasciculata
Cryptosporidium
parvum
Entamoeba histolytica
Giardia lamblia
Kentrophoros sp.
Leishmania aethiopica
Leishmania amazonensis
Leishmania braziliensis
Leishmania donovani
Leishmania infantum
Leishmania enriettii
Leishmania gerbilli
Leishmania guyanensis
Leishmania hertigi
Leishmania major
Leishmania mexicana
Leishmania tarentolae
Leishmania tropica
Neospora caninum
Onchocerca volvulus
Plasmodium berghei
Plasmodium falciparum
Plasmodium knowlesi
Porphyra purpurea
Toxoplasma gondii
Treponema pallidum
Trichomonas vaginalis
CA 02307010 2000-OS-19
Table 5. Antibiotic resistance genes selected for diagnostic purposes.
Genes Antibiotics Bacteriai ACCESSION NO. SEA ID NO.
(genes)
aac(3)-Ib AminoglycosidesEnterobacteriaceaeL06157
2
Pseudomonads
aac(3)-llb AminoglycosidesEnterobacteriaceae,M97172
2
Pseudomonads
aac(3)-IVa AminoglycosidesEnterobacteriaceaeX01385
2
aac(3)-Vla AminoglycosidesEnterobacteriaceae,M88012
2
Pseudomonads
aac(2)-la AminoglycosidesEnterobacteriaceae,X04555
2
Pseudomonads
83-86
3
1 aac(6)-aph(2')AminoglycosidesEnterococcus sp.,
S 2
Staphylococcus
sp.
aac(6)-la, AminoglycosidesEnterobacteriaceae,M18967
2
Pseudomonads
aac(6)-lc AminoglycosidesEnterobacteriaceae,M94066
2
Pseudomonads
112 4
aac(6)-lla AminoglycosidesPseudomonads 53-54
2 3
aad8 [ant(2")-laAminoglycosidesEnterobacteriaceae 55-56
2] 3
aacCl [aac(3)-laAminoglycosidesPseudomonads 57-58
2] 3
aacC2 [aac(3)-IlaAminoglycosidesPseudomonads 59-60
2] 3
aacC3 [aac(3)-IllAminoglycosidesPseudomonads 65-66
2] 3
aacA4 [aac(6)-IbAminoglycosidesPseudomonads
2]
ant(3')-la AminoglycosidesEnterobacteriaceae,X02340
2
Enterococcus sp.,M10241
Staphylococcus
sp.
ant(4)-la AminoglycosidesStaphylococcus V01282
2 sp.
aph(3)-la AminoglycosidesEnterobacteriaceae,J01839
2
Pseudomonads
aph(3)-Ila AminoglycosidesEnterobacteriaceae,V00618
2
Pseudomonads
aph(3)-Illa AminoglycosidesEnterococcus sp.,V01547
2
Staphylococcus
sp.
aph(3')-Vla AminoglycosidesEnterobacteriaceae,X07753
2
Pseudomonads
rrs 2 Streptomycin M. tuberculosis L15307
S62531
rpsL 2 Streptomycin M. tuberculosis, X80120
M. avium complex U14749
X70995
L08011
110 4
bla pxq 5'6 f3-lactams Enterobacteriaceae,
Pseudomonads
45-48
3
blaRpg 5 f3-lactams Haemophilus sp.
Pasteurella sp.
41-44
3
bIaSHV 5'6 f3-lactams Enterobacteriacea,
Pseudomonas aeruginosa
37-40
3
bla7-EM 5'6 (3-lactams Enterobacteriaceae,
Neisseria sp.,
Haemophilus sp.
bIaCARg f3-lactams Pseudomodas sp., J05162
5
Enterobacteriaceae S46063
M69058
71
CA 02307010 2000-OS-19
Table 5. Antibiotic resistance genes selected for diagnostic purposes
(continued).
Antibiotics Bacterial ACCESSION SEO ID NO.
NO.
Genes (genes)
bIaCTX-M-15t3-lactams Enterobacteriaceae X92506
bIaCTX-M-2 fi-lactams Enterobacteriaceae X92507
5
blaCMy_27 f3-lactams Enterobacteriaceae X91840
blapER-15 f3-lactams Enterobacteriaceae, 221957
Pseudomodanaceae
bIapER-27 f3-lactams Enterobacteriaceae X93314
blalMp 5 f3-lactams Enterobacteriaceae, AJ223604
Pseudomonas aeruginosa
111 4
blaZ 12 f3-lactams Enterococcus sp.,
Staphylococcus sp.
97-98 3
mecA 12 f3-lactams Staphylococcus sp.
penA 13 f3-lactams Neisseria gonorrhoeaeX54021
pbpla 13 f3-lactams Streptococcus pneumoniae SEE TABLE
7
M90527
X67872
AB006868
AB006874
X67873
AB006878
AB006875
AB006877
AB006879
AF046237
AF046235
AF026431
AF046232
AF046233
AF046236
X67871
3 249095
5
AF046234
AB006873
X67866
X67868
AB006870
AB006869
AB006872
X67870
AB006871
xs78s7
X67869
AB006876
AF046230
AF046238
249094
pbp2b 13 f3-lactams Streptococcus pneumoniae SEE TABLE
7
X16022
M25516
M25518
M25515
020071
020084
020082
020067
020079
222185
020072
72
CA 02307010 2000-OS-19
Table 5. Antibiotic resistance genes selected for diagnostic purposes
(continued).
Bacterial ACCESSION SEO ID NO.
NO.
Genes Antibiotics (genes)
pbp2b 13 f3-lactams Streptococcus 020083
pneumoniae
020081
M25522
020075
020070
020077
020068
222184
020069
u2oo78
M25521
M25525
M25519
221981
M25523
M25526
M25524
222230
020073
u2ooao
020074
020076
M25520
M25517
pbp2x 13 f3-lactams Streptococcus SEE TABLE
pneumoniae 7
X16367
X65135
AB011204
AB011209
AB011199
AB011200
AB011201
AB011202
AB011198
AB011208
AB011205
AB015852
AB011210
AB015849
AB015850
AB015851
AB015847
AB015846
AB011207
AB015848
249096
99-102 3
int [3 -lactams, Enterobacteriaceae,
trimethoprim
103-106 3
sul aminoglycosides,Pseudomonads
antiseptic,
chloramphenicol
113 4
ermA 14 Macrolides, Staphylococcus
sp.
lincosamides,
streptogramin
B 114 4
erm8 14 Macrolides, Enterobacteriaceae,
Staphylococcus
sp.
lincosamides, Enterococcus sp.
streptogramin Streptococcus
B sp.
73
CA 02307010 2000-OS-19
tibiotic resistance
genes selected
for diagnostic
purposes (continued).
A
Table 5. n
Antibiotics Bacterial ACCESSION SEO ID
NO. NO.
Genes (genes)
115 4
ermC 14 Macrolides, Enterobacteriaceae,
lincosamides, Staphylococcus
sp.
streptogramin
B
ereA 12 Macrolides Enterobacteriaceae,M11277
Staphylococcus
sp.
ere812 Macrolides EnterobacteriaceaeA15097
Staphylococcus
sp. 77_g0
3
msrA 12 Macrolides Staphylococcus
sp.
mtr 8 Macrolides Neisseria gonorrhoeaeS42418
S40252
S42417
S40251
225796
U 14993
051007
6151006
051073
AF037040
AF037041
mefE 8 Macrolides Streptococcus U70055
mefA sp.
, U83667
mphA 8 Macrolides Enterobacteriaceae,D16251
Staphylococcus
sp.
IinAllinA'9Lincosamides Staphylococcus J03947
sp.
M14039
IinB 10 Lincosamides Enterococcus faeciumAF110130
rrna 11 Macrolides Mycobacterium U74494
avium complex
89-90
3
vga 15 Streptrogramin Staphylococcus
sp.
vgb 15 Streptrogramin Staphylococcus M36022
sp.
87-88
3
vat 15 Streptrogramin Staphylococcus
sp.
vatB 15 Streptrogramin Staphylococcus U19456
sp.
L38809
satA 15 Streptrogramin Enterococcus faecium 81-82
3
ileS 12 Mupirocin Staphylococcus X74219
aureus
mupA 12 Mupirocin Staphylococcus X75439
aureus
gyrA 16 Quinolones Gram-positive X95718
and
gram-negative X06744
bacteria
X57174
X16817
X71437
AF065152
AF060881
D32252
parClgrlA Quinolones Gram-positive AB005036
16 and
gram-negative AF056287
bacteria
X95717
5 AF 129764
5
AB017811
AF065152
74
CA 02307010 2000-OS-19
Table 5. Antibiotic resistance genes selected for diagnostic purposes
(continued).
Genes Antibiotics Bacterial ACCESSION NO. SEQ ID NO.
Inanasl
parElgrlB Quinolones Gram-positive bacteriaX95717
16
AF065153
AF058920
norA 16 Quinolones Staphylococcus sp. D90119
M80252
M97169
mexR (nal8)Quinolones Pseudomonas aeruginosa023763
16
nfx816 Quinolones Pseudomonas aeruginosaX65646
cat 12 ChloramphenicolGram-positive and M55620
gram-negative bacteriaX15100
A24651
M28717
A00568
A00569
X74948
Y00723
A24362
A00569
M93113
M62822
M58516
rpo817 Rifampin Mycobacterium tuberculosisAF055891
AF055892
S71246
L27989
AF055893
inhA 17 Isoniazid Mycobacterium tuberculosisAF106077
002492
katG 17 Isoniazid Mycobacterium tuberculosis040593
006259
U 06260
006261
006262
040594
040595
ahpC 17 Isoniazid Mycobacterium tuberculosis043812
057761
024085
016243
U 58030
018264
emb817 Ethambutol Mycobacterium tuberculosis068480
pncA 17 PyrazinamideMycobacterium tuberculosis059967
67-70 3
vanA 12 Vancomycin Enterococcus sp. SEE TABLE
7
116 4
van812 Vancomycin Enterococcus sp. 117 4
vanCl 12 Vancomycin Enterococcus gallinarum SEE TABLE
7
vanC212 Vancomycin Enferococcus casseliflavus SEE TABLE
7
094521
094522
094523
094524
094525
L29638
CA 02307010 2000-OS-19
Table 5. Antibiotic resistance genes selected for diagnostic purposes
(continued).
Genes Antibiotics Bacterial ACCESSION NO. SEO ID NO.
lnanasl
vanC312 Vancomycin Enterococcus flavescensSEE TABLE 7
L29639
U72706
L29640
vanD 18 Vancomycin Enterococcus faeciumAF130997
tetB 19 Tetracycline Gram-negative bacteriaJ01830
tetM 19 Tetracycline Gram-negative and X52632
Gram-positive bacteria
su11120 Sulfonamides Gram-negative bacteriaD37827
M36657
AF017389
AF017391
dhfrla Trimethoprim Gram-negative bacteriaAJ238350
X17477
20 dhfrlb Trimethoprim Gram-negative bacteria250805
20
250804
dhfrV 20 Trimethoprim Gram-negative bacteriaX12868
dhfrV1120 Trimethoprim Gram-negative bacteriaU31119
dhfrV11120Trimethoprim Gram-negative bacteriaU10186
dhfrlX Trimethoprim Gram-negative bacteriaX57730
20
dhfrX1120 Trimethoprim Gram-negative bacteria221672
dfrA 20 Trimethoprim Staphylococcus sp. AF045472
U40259
AF051916
1 Bacteria having high incidence for the specified antibiotic resistance gene.
The presence of the antibiotic
resistance genes in other bacteria is not excluded.
2 Shaw, K. J., P. N. Rather, R. S. Hare, and G. H. Miller. 1993. Molecular
genetics of aminoglycoside
resistance genes and familial relationships of the aminoglycoside-modifying
enzymes. Microbiol. Rev.
57:138-163.
3 Antibiotic resistance genes from our co-pending US patent no. 6,001,564 for
which we have selected
PCR primer pairs.
4 These SEQ ID NOs. refer to a previous patent (application W098/20157).
5 Bush, K., G.A. Jacoby and A. Medeiros. 1995. A functional classification
scheme for f3-lactamase and its
correlation with molecular structure. Antimicrob. Agents. Chemother. 39:1211-
1233.
6 Nucleotide mutations in bIaSHU bIaTEM, and blapXq, are associated with
extended-spectrum f3-
lactamase or inhibitor-resistant f3-lactamase.
7 Bauerfeind, A., Y. Chong, and K. Lee. 1998. Plasmid-encoded AmpC beta-
lactamases: how far have we
gone 10 ears after discovery? Yonsei Med. J. 39:520-525.
8 Sutcliffe, J., T. Grebe, A. Tait-Kamradt, and L. Wondrack. 1996. Detection
of erythromycin-resistant
determinants by PCR. Antimicrob. Agent Chemother. 40:2562-2566.
9 Leclerc, R., A., Brisson-Noel, J. Duval, and P. Courvalin. 1991. Phenotypic
expression and genetic
heterogeneity of lincosamide inactivation in Staphylococcus sp. Antimicrob.
Agents. Chemother. 31:1887-
1891.
10 Bozdogan, B., L. Berrezouga, M.-S. Kuo, D. A. Yurek, K. A. Farley, B. J.
Stockman, and R. Leclercq.
1999. A new gene, IinB, conferring resistance to lincosamides by
nucleotidylation in Enterococcus
faecium HM1025. Antimicrob. Agents. Chemother. 43:925-929.
11 Cockerill III, F.R. 1999. Genetic methods for assessing antimicrobial
resistance. Antimicrob. Agents.
Chemother. 43:199-212
12 Tenover, F. C., T. Popovic, and O Olsvik. 1996. Genetic methods for
detecting antibacterial resistance
genes. pp. 1368-1378. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C.
Tenover, R. H. Yolken (eds).
Manual of clinical microbiology. 6th ed., ASM Press, Washington, D.C. USA
13 powson, C. G., T. J. Tracey, and B. G. Spratt. 1994. Origin and molecular
epidemiology of penicillin-
76
CA 02307010 2000-OS-19
binding-protein-mediated resistance to f3-lactam antibiotics. Trends Molec.
Microbiol.2: 361-366.
14 Jensen, L. B., N. Frimodt-Moller, F. M. Aarestrup. 1999. Presence of erm
gene classes in Gram-positive
bacteria of animal and human origin in Denmark. FEMS Microbiol. 170:151-158.
15 Thal, L. A., and M. J. Zervos. 1999. Occurrence and epidemiology of
resistance to virginimycin and
streptrogramins. J. Antimicrob. Chemother. 43:171-176'
16 Martinez J. L., A. Alonso, J. M. Gomez-Gomez, and F. Baquero. 1998.
Quinolone resistance by mutations
in chromosomal gyrase genes. Just the tip of the iceberg? J. Antimicrob.
Chemother. 42:683-688
17 Cockerill III, F.R. 1999. Genetic methods for assessing antimicrobial
resistance. Antimicrob. Agents.
Chemother. 43:199-212.
18 Casadewall, B. and P. Courvalin. 1999 Characterization of the vanD
glycopeptide resistance gene cluster
from Enterococcus faecium BM 4339. J. Bacteriol. 181:3644-3648.
19 Roberts, M.C. 1999. Genetic mobility and distribution of tetracycline
resistance determinants.Ciba Found.
Symp. 207:206-222.
Huovinen, P., L. Sundstrom, G. Swedberg and O. Skold. 1995. Trimethoprim and
sulfonamide
15 resistance.Antimicrob. Agent Chemother. 39:279-289.
77
CA 02307010 2000-OS-19
Table 6. List of bacterial toxins selected for diagnostic purposes.
Toxin Accession number
Organism
Actinobacillus actinomycetemcomitansCytolethal distending toxinAF006830
(cdtA, cdlB, cdtC)
Leukotoxin (ItxA) M27399
Actinomyces pyogenes Hemolysin (pyolysin) U84782
Aeromonas hydrophila Aerolysin (aerA) M16495
Haemolysin (hlyA) U81555
Cytotonic enterotoxin (alt)L77573
Bacillus anthracis Anthrax toxin (cya) M23179
Bacillus cereus Enterotoxin (bceT) D17312
AF192766,AF192767
Enterotoxic hemolysin BL AJ237785
15 Non-haemolytic enterotoxinsY19005
A,B and C (nhe)
Bacillus mycoides Hemolytic enterotoxin HBL AJ243150 to
AJ243153
Bacillus pseudomycoides Hemolytic enterotoxin HBL AJ243154 to
AJ243156
Bacteroides fragilis Enterotoxin (bftP) U67735
Matrix metalloprotease/enterotoxinS75941, AF038459
(fragilysin)
Metalloprotease toxin-2 U90931
AF081785
Metalloprotease toxin-3 AF056297
Bordetella bronchisepticaAdenylate cyclase hemolysin237112, U22953
(cyaA)
Dermonecrotic toxin (dnt) U59687
25 AB020025
Bordetella pertussis Pertussis toxin (S1 subunit,AJ006151
tox)
AJ006153
Patents: AJ006155
EP0322533-A 2 05ju189 AJ006157
EP0322115-A 5 28jun89 AJ006159
EP0396964-A 1 l4nov90 AJ007363
JP1987228286-A 1 7oct87 M14378, M16494
AJ007364
M13223
35 X16347
Adenyl cyclase (cya) 18323
Dermonecrotic toxin (dnt) U10527
Campylobacter jejuni Cytolethal distending toxinU51121
~ (cdtA, cdlB, cdtC)
Citrobacter freundii Shiga-like toxin (slt-IIcA)X67514, S53206
Clostridium botulinum Botulism toxin (BoNT) X52066, X52088
X73423
The A,B,E and F serotypes M30196
are
neurotoxic for humans X70814
X70819
45 The other serotypes have X71343
not been considered
211934
Partial sequences (<200 X70817
bp) have
not been considered M81186
X70818
5o X70815
X62089
X62683
S76749
X81714
55 x7os1 s
x7os2o
X70281
L35496
M92906
Clostridium difficile A toxin (enterotoxin) (tcdA)AB012304
AF053400
Y12616
X51797
X17194
65 M30307
78
CA 02307010 2000-OS-19
Table 6. List of bacterial toxins selected for diagnostic purposes
(continued).
Organism Toxin Accession
number
Clostridium difficile B toxin (cytotoxin) (toxB)223277
X53138
Clostridium perfringens Alpha (phospholipase C) L43545
(cpa)
L43546
L43547
L43548
X13608
X17300
D10248
Beta (dermonecrotic protein)L13198
(cpb)
X83275
I S L77965
Enterotoxin (cpe) AJ000766
M98037
X81849
X71844
Y16009
Enterotoxin pseudogene AF037328
(not expressed)
AF037329
AF037330
Epsilon toxin (etxD) M80837
25 M95206
X60694
Iota (la and Ib) X73562
Lambda (metalloprotease) D45904
Theta (perfringolysin M36704
O)
Clostridium sordellii Cytotoxin L X82638
Clostridium tetani Tetanos toxin X06214
X04436
Corynebacterium diphtheriaeDiphtheriae toxin X00703
Patent:
3 5 J P 1985227681-A/1
Corynebacterium pseudotuberculosisPhospholipase C A21336
Patent: WO 9011351-A 2
Eikenella corrodens lysine decarboxylase (cadA)U89166
Enterobacter cloacae Shiga-like toxin II 250754, U33502
Enterococcus faecalis Cytolysin B (cyll3) M38052
Escherichia coli (EHEC) Hemolysin toxin (hlyA AF043471
and ehxA)
X94129
X79839
X86087
45 AB011549
AF074613
Shiga-like (Vero cytotoxin)X81418
(stx)
M14107
Contains the sequences M10133
for both the
A and B subunits M12863
Patent:JP 1995008280-A/1 X81417
X81416
X81415
236900
55 L11078
L04539
L11079
X65949
M21534
M29153
237725
236901
X61283
AB017524
65 u72191
79
CA 02307010 2000-OS-19
Table 6. List of bacterial toxins selected for diagnostic purposes
(continued).
Toxin Accession nun
Organism
Escherichia coli (ETEC)Enterotoxin (heat-labile) M17874
(eltB)
Patents: EP 0145486-A 5 M17873
WO 9313202-A 42 J01605
JP 1986005097-A AB011677
JP 1992320675-A
Enterotoxin (heat-stable) L11241
(astA) (estAi)
M58746
M29255
V00612
J01831
Escherichia coli (other)Cytolethal-distending toxin003293
15 (cdt) (3 genes) 004208
089305
Cytotoxic necrotizing factor042629
1 (cnfl )
Microcin 24 (mtlS) 047048
Autotransporter enterotoxinAF056581
(Pet) (cytotoxin)
20 Haemophilus ducreyi Cytolethal distending toxin053215
(cdtA, cdtB, cdtC)
Helicobacter pylori Vacuolating toxin (vacA) 007145
080067
080068
AF077938
25 AF077939
AF077940
AF077941
Legionella pneumophila Structural toxin protein AF057703
(rtxA)
Listeria monocytogenes Listeriolysin O (IisA, hlyA)X15127
3o M24199
X60035
025452
025443
025446
35 025449
Pasteurella multocida Mitogenic toxin (dermonecroticX57775,
toxin) 228388
X51512
X52478
Proteus mirabilis Hemolysin (hpmA) M30186
40 Pseudomonas aeruginosa Cytotoxin (Enterotoxin A) X14956
Salmonella typhimurium Calmodulin-sensitive adenylate
cyalase toxin (cya) AF060869
Cytolysin (salmolysin) (slyA)003842
Enterotoxin (stn) L16014
Serratia marcescens Hemolysin (shlA) M22618
4.5 Shigella dysenteriae Shiga toxin (2 subunits) X07903 M32511
type 1
(stxA and stxB) M19437
M24352,
M21947
Shigella flexneri ShET2 enterotoxin (senA) 254211
247381
Enterotoxin 1 (sell A and 035656
sell B)
Hemolysin E (hlyE, clyA, AF200955
sheA)
Shigella sonnei Shiga toxin (2 subunits) AJ132761
(stxA and stxB)
Sphingomonas paucimobilisBeta-hemolysin (hlyA) L01270
Staphylococcus aureus Gamma-hemolysin (hlg2) D42143
5 L01055
Enterotoxin 093688
Enterotoxin A (sea) L22565,
L22566
M18970
Enterotoxin B M11118
Enterotoxin C1 (entC1 ) X05815
Enterotoxin C2 (entC2) P34071
Enterotoxin C3 (enlC3) X51661
Enterotoxin D (secQ M94872
Enterotoxin E M21319
80
CA 02307010 2000-OS-19
Table 6. List of bacterial toxins selected for diagnostic purposes
(continued).
Toxin Accession number
Organism
Staphylococcus aureus Enterotoxin G (seg) AF064773
Enterotoxin H (seh) 011702
Enterotoxin I (sei) AF064774
Enterotoxin J AF053140
Exfoliative toxin A (ETA) (Epidermolytic toxin A) M17347
M17357
L25372,
M20371
to Exfoliative toxin B (ETB) M17348,
M13775
Leukocidin R (F and S component, IukF and IukS) X64389,
S53213
(Hemolysin B and C) X72700
L01055
I S Toxic shock syndrome toxin 1 (TSST-1 ) X01645
(alpha toxin) M90536
(alpha hemolysin) J02615
093688
Staphylococcus epidermidis Delta toxin (hlcn AF068634
Staphylococcus intermedius Enterotoxin 1 091526
Leukocidin R (F and S component, IukF and IukS) X79188
(synergohymenotropic toxin)
Streptococcus pneumoniaePneumolysin X52474
25 Streptococcus pyogenesStreptococcus pyrogenic X61560
exotoxin A (speA)
(and 19 others)
X03929
040453, M19350
Pyrogenic exotoxin B (spell)063134
3o M86905, M35110
Vibrio cholerae Cholerae toxin (ctxA and X00171
ctxB subunits)
Patents: X76390
JP 1995008279-A 1 X58786
EP 0368819-A 12 (ctxB) X58785, S55782
35 WO 9313202-A 45 (ctxA) D30052
D30053
K02679
AF175708
Accessory cholera enterotoxin222569, AF175708
(ace)
Heat-stable enterotoxin X74108, M85198
(sfo)
M97591, L03220
Zonula occludens toxin (zot)M83563, AF175708
Vibrio parahaemolyticusThermostable direct hemolysinS67841
(fdh)
Vibrio vulnificus Cytolysin (whA) M34670
45 Yersinia enterocoliticaHeat-stable enterotoxin 009235, X65999
(yst)
Heat-stable enterotoxin D88145
type B (ystB)
Heat-stable enterotoxin D63578
type C (ystC)
Yersinia kristensenii Enterotoxin X69218
Yersinia pestis Toxin X92727
81
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing.
SE(~ ID NO. Bacterial,fungal or parasitical species Source Comments*
1 Acinetobacter baumannii This patent tuf
t
f
2 Actinomyces meyeri This patent u
tuf
3 Aerococcus viridans This patent
4 Achromobacterxylosoxidans subsp. This patent tuf
denitrificans
Anaerorhabdus furcosus This patent tuf
6 Bacillus anthracis This patent tuf
t
f
7 Bacillus cereus This patent u
f
t
g Bacteroides distasonis This patent u
t
f
g Enterococcus casseliflavus This patent u
t
f
10 Staphylococcus saprophyticus This patent u
t
f
11 Bacteroides ovatus This patent u
t
f
12 Bartonella henselae This patent u
f
t
13 Bifidobacterium adolescentis This patent u
f
t
14 Bifidobacterium dentium This patent u
t
f
15 Brucella abortus This patent u
tuf
16 Burkholderia cepacia This patent tuf
17 Cedecea davisae This patent tuf
1 g Cedecea neteri This patent f
t
19 Cedecea lapagei This patent u
f
t
20 Chlamydia pneumoniae This patent u
f
t
21 Chlamydia psittaci This patent u
f
t
22 Chlamydia trachomatis This patent u
f
t
23 Chryseobacterium meningosepticum This patent u
f
t
24 Citrobacteramalonaticus This patent u
25 Citrobacter braakii This patent tuf
26 Citrobacter koseri This patent tuf
f
t
27 Citrobacter farmeri This patent u
f
t
2g Citrobacter freundii This patent u
29 Citrobactersedlakii This patent tuf
30 Citrobacter werkmanii This patent tuf
31 Citrobacter youngae This patent tuf
32 Clostridium perfringens This patent tuf
33 Comamonas acidovorans This patent tuf
34 Corynebacterium bovis This patent tuf
35 Corynebacterium cervicis This patent tuf
36 Corynebacterium flavescens This patent tuf
37 Corynebacterium kutscheri This patent tuf
38 Corynebacterium minutissimum This patent tuf
f
t
39 Corynebacterium mycetoides This patent u
40 Corynebacterium pseudogenitalium This patent tuf
41 Corynebacterium renale This patent tuf
42 Corynebacterium ulcerans This patent tuf
43 Corynebacterium urealyticum This patent tuf
44 Corynebacterium xerosis This patent tuf
45 Coxiella burnetii This patent tuf
46 Edwardsiella hoshinae This patent tuf
47 Edwardsiella tarda This patent tuf
48 Eikenella corrodens This patent tuf
49 Enterobacter aerogenes This patent tuf
f
t
50 Enterobacter agglomerans This patent u
tuf
51 Enterobacter amnigenus This patent f
t
52 Enterobacter asburiae This patent u
53 Enterobacter cancerogenus This patent tuf
f
t
54 Enterobacter cloacae This patent u
tuf
55 Enterobacter gergoviae This patent tuf
56 Enterobacter hormaechei This patent
57 Enterobacter sakazakii This patent tuf
58 Enterococcus casseliflavus This patent tuf
5g Enterococcus cecorum This patent tuf
f
t
60 Enterococcus dispar This patent u
f
f
61 Enterococcus durans This patent u
82
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEQ ID NO. Bacterial,fungal or parasitical species Source Comments*
$ 62 Enterococcus faecalis This patent tuf
63 Enterococcus faecalis This patent tuf
64 Enterococcus faecium This patent tuf
t
t
65 Enterococcus flavescens This patent u
tuf
66 Enterococcus gallinarum This patent f
t
67 Enterococcus hirae This patent u
t
f
6g Enterococcus mundtii This patent u
t
f
6g Enterococcus pseudoavium This patent u
t
f
70 Enterococcus raffinosus This patent u
t
f
71 Enterococcus saccharolyticus This patent u
t
f
72 Enterococcus solitarius This patent u
t tuf (C)
t
i
73 Enterococcus casseliflavus en nknown
s pa
Th
74 Staphylococcus saprophyticus This patent u
t tuf (C)
t
i
75 Enterococcus flavescens en tuf (C)
s pa
Th
t
t
hi
76 Enterococcus gallinarum en t
s pa f
T
77 Ehrlichia canis This patent u
tuf
7g Escherichia coli This patent t
t
7g Escherichia fergusonii This patent u
t
f
g0 Escherichia hermannii This patent u
tuf
g1 Escherichia vulneris This patent
82 Eubacterium lentum This patent tuf
g3 Eubacterium nodatum This patent tuf
84 Ewingella americana This patent tuf
g5 Francisella tularensis This patent tuf
86 Fusobacterium nucleatum subsp. This patent tuf
polymorphum
87 Gemella haemolysans This patent tuf
gg Gemella morbillorum This patent tuf
f
t
gg Haemophilus actinomycetemcomitans This patent u
90 Haemophilus aphrophilus This patent tuf
91 Haemophilus ducreyi This patent tuf
92 Haemophilus haemolyticus This patent tuf
93 Haemophilus parahaemolyticus This patent tuf
94 Haemophilus parainfluenzae This patent tuf
95 Haemophilus paraphrophilus This patent tuf
g6 Haemophilus segnis This patent tuf
97 Hafnia alvei This patent tuf
gg Kingella kingae This patent tuf
gg Klebsiella omithinolytica This patent tuf
100 Klebsiella oxytoca This patent tuf
f
t
101 Klebsiella planticola This patent u
102 Klebsiella pneumoniae subsp. ozaenaeThis patent tuf
103 Klebsiella pneumoniae pneumoniae This patent tuf
104 Klebsiella pneumoniae subsp. rhinoscleromatisThis patent tuf
105 Kluyvera ascorbata This patent tuf
106 Kluyvera cryocrescens This patent tuf
f
t
107 Kluyvera georgiana This patent u
f
t
108 Lactobacillus casei subsp. casei This patent u
f
t
109 Lactococcus lactic subsp. lactic This patent u
110 Leclercia adecarboxylata This patent tuf
111 Legionella micdadei This patent tuf
112 Legionella pneumophila subsp. pneumophilaThis patent tuf
113 Leminorella grimontii This patent tuf
114 Leminorella richardii This patent tut
115 Leptospira interrogans This patent tuf
t
f
116 Megamonas hypermegale This patent u
f
t
117 Mitsuokella multacidus This patent u
f
t
118 Mobiluncus curtisii subsp. holmesiiThis patent u
119 Moellerella wisconsensis This patent tut
t
f
120 Moraxella catarrhalis This patent u
tut
121 Morganella morganii subsp. morganiiThis patent
122 Mycobacterium tuberculosis This patent tuf
83
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEO ID NO. Bacterial,fungal or parasitical species Source Comments*
123 Neisseria cinerea This patent tuf
t
f
124 Neisseria elongata subsp. elongataThis patent u
fuf
125 Neisseria flavescens This patent t
f
126 Neisseria gonorrhoeae This patent u
tuf
127 Neisseria lactamica This patent tuf
128 Neisseria meningitidis This patent t
f
12g Neisseria mucosa This patent u
tuf
130 Neisseria sicca This patent t
f
131 Neisseria subflava This patent u
t
f
132 Neisseria weaveri This patent u
f
t
133 Ochrobactrum anthropi This patent u
t
f
134 Pantoea agglomerans This patent u
tuf
135 Pantoea dispersa This patent f
t
136 Pasteurella multocida This patent u
f
t
137 Peptostreptococcus anaerobius This patent u
138 Peptostreptococcus asaccharolyticusThis patent tuf
139 Peptostreptococcus prevotii This patent tuf
f
t
140 Porphyromonas asaccharolytica This patent u
tuf
141 Porphyromonas gingivalis This patent tuf
142 Pragia fontium This patent f
t
143 Prevotella melaninogenica This patent u
t
f
144 Prevotella oralis This patent u
tuf
145 Propionibacterium acnes This patent tuf
146 Proteus mirabilis This patent tuf
147 Proteus penneri This patent t
f
148 Proteus vulgaris This patent u
tuf
149 Providencia alcalifaciens This patent f
t
150 Providencia rettgeri This patent u
151 Providencia rustigianii This patent tuf
f
t
152 Providencia stuartii This patent u
f
t
153 Pseudomonas aeruginosa This patent u
f
t
154 Pseudomonas fluorescens This patent u
f
t
155 Pseudomonas stutzeri This patent u
f
t
156 Psychrobacter phenylpyruvicum This patent u
t
t
157 Rahnella aquatilis This patent u
f
t
158 Salmonella choleraesuis subsp.arizonaeThis patent u
159 Salmonella choleraesuis subsp. This patent tuf
choleraesuis
serotype Choleraesuis
lmonella choleraesuis subsp. diarizonaeThis patent tuf
S
160 a This patent tuf
161 Salmonella choleraesuis subsp.
choleraesuis
serotype Heidelberg
This patent tuf
162 Salmonella choleraesuis subsp. t
houtenae f
163 Salmonella choleraesuis subsp. This patent u
indica
164 Salmonella choleraesuis subsp. This patent tuf
salamae
165 Salmonella choleraesuis subsp. tuf
choleraesuis serotype Typhi This
patent
166 Serratia fonticola This patent tuf
f
t
167 Serratia liquefaciens This patent u
tuf
168 Serratia marcescens This patent t
f
169 Serratia odorifera This patent u
tuf
170 Serraiia plymuthica This patent tuf
171 Serratia rubidaea This patent f
t
172 Shigella boydii This patent u
t
f
173 Shigella dysenteriae This patent u
t
f
174 Shigella flexneri This patent u
tuf
175 Shigella sonnei This patent tuf
176 Staphylococcus aureus This patent f
t
177 Staphylococcus aureus This patent u
tuf
178 Staphylococcus aureus This patent t
f
17g Staphylococcus aureus This patent u
t
f
180 Staphylococcus aureus subsp. aureusThis patent u
tuf
181 Staphylococcus auricularis This patent tuf
182 Staphylococcus capitis subsp. This patent
capitis
84
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEQ ID NO. Bacterial,fungal or parasitical species Source Comments*
183 Macrococcus caseolyticus This patent tuf
t
f
184 Staphylococcus cohnii This patent u
f
t
185 Staphylococcus epidermidis This patent u
f
t
186 Staphylococcus haemolyticus This patent u
tuf
187 Staphylococcus warneri This patent t
f
188 Staphylococcus haemolyticus This patent u
tuf
189 Staphylococcus haemolyticus This patent f
t
190 Staphylococcus haemolyticus This patent u
t
f
191 Staphylococcus hominis subsp. hominisThis patent u
192 Staphylococcus warneri This patent tuf
tuf
193 Staphylococcus hominis This patent tuf
194 Staphylococcus hominis This patent f
t
195 Staphylococcus hominis This patent u
t
f
196 Staphylococcus hominis This patent u
t
f
1 g7 Staphylococcus lugdunensis This patent u
tuf
198 Staphylococcus saprophyticus This patent t
t
1 gg Staphylococcus saprophyticus This patent u
f
t
200 Staphylococcus saprophyticus This patent u
f
t
201 Staphylococcus sciuri subsp. sciuriThis patent u
t
f
202 Staphylococcus warneri This patent u
t
f
203 Staphylococcus warneri This patent u
t
f
204 Bifidobacterium longum This patent u
t
f
205 Stenotrophomonas maltophilia This patent u
tuf
206 Streptococcus acidominimus This patent f
t
207 Streptococcus agalactiae This patent u
t
t
208 Streptococcus agalactiae This patent u
tuf
209 Streptococcus agalactiae This patent f
t
210 Streptococcus agalactiae This patent u
f
t
211 Streptococcus anginosus This patent u
f
t
212 Streptococcus bovis This patent u
f
t
213 Streptococcus anginosus This patent u
f
t
214 Streptococcus cricetus This patent u
tuf
215 Streptococcus cristatus This patent f
t
216 Streptococcus downei This patent u
f
t
217 Streptococcus dysgalactiae This patent u
t
f
218 Streptococcus equi subsp. equi This patent u
t
t
219 Streptococcus ferus This patent u
tuf
220 Streptococcus gordonii This patent f
t
221 Streptococcus anginosus This patent u
t
f
222 Streptococcus macacae This patent u
t
f
223 Streptococcus gordonii This patent u
tuf
224 Streptococcus mutans This patent tuf
225 Streptococcus parasanguinis This patent f
t
226 Streptococcus ratti This patent u
f
t
227 Streptococcus sanguinis This patent u
tuf
228 Streptococcus sobrinus This patent t
f
229 Streptococcus suis This patent u
tuf
230 Streptococcus uberis This patent f
t
231 Streptococcus vestibularis This patent u
t
f
232 Tatumella ptyseos This patent u
tuf
233 Trabulsiella guamensis This patent f
t
234 Veillonella parvula This patent u
f
t
235 Yersinia enterocolitica This patent u
tuf
236 Yersinia frederiksenii This patent f
t
237 Yersinia intermedia This patent u
t
f
238 Yersinia pestis This patent u
f
t
239 Yersinia pseudotuberculosis This patent u
240 Yersinia rohdei This patent tuf
241 Yokenella regensburgei This patent tuf
t D
t at
hi
242 Achromobacterxylosoxidans subsp. en p
denitrificans s pa
T
243 Acinetobacterbaumannii This patent atpD
t D
t at
Thi
244 Acinetobacter Iwoffii en p
s pa
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEQ ID NO. Bacterial,fungal or parasitical species Source Comments*
245 Staphylococcus saprophyticus This patent atpD
246 Alcaligenes faecalis This patent atpD
247 Bacillus anthracis This patent atpD
248 Bacillus cereus This patent atpD
249 Bacteroides distasonis This patent atpD
250 Bacteroides ovatus This patent atpD
251 Leclercia adecarboxylata This patent atpD
252 Stenotrophomonas maltophilia This patent atpD
253 Bartonella henselae This patent atpD
254 Bifidobacterium adolescentis This patent atpD
255 Brucella abortus This patent atpD
256 Cedecea davisae This patent atpD
257 Cedecea lapagei This patent atpD
258 Cedecea neteri This patent atpD
259 Chryseobacterium meningosepticum This patent atpD
260 Citrobacter amalonaticus This patent atpD
261 Citrobacter braakii This patent atpD
262 Citrobacter koseri This patent atpD
263 Citrobacter farmeri This patent atpD
264 Citrobacter freundii This patent atpD
265 Citrobacter koseri This patent atpD
266 Citrobacter sedlakii This patent atpD
267 Citrobacter werkmanii This patent atpD
268 Citrobacter youngae This patent atpD
269 Clostridium innocuum This patent atpD
t D
t at
hi
270 Clostridium perfringens en p
s pa D
T at
t
t
Thi
272 Corynebacterium diphtheriae en p
s pa
273 Corynebacterium pseudodiphtheriticumThis patent atpD
274 Corynebacterium ulcerans This patent atpD
275 Corynebacterium urealyticum This patent atpD
276 Coxiella burnetii This patent atpD
277 Edwardsiella hoshinae This patent atpD
278 Edwardsiella tarda This patent atpD
27g Eikenella corrodens This patent atpD
280 Enterobacter agglomerans This patent atpD
281 Enterobacter amnigenus This patent atpD
282 Enterobacter asburiae This patent atpD
283 Enterobacter cancerogenus This patent atpD
t D
t at
Thi
284 Enterobacter cloacae en p
s pa D
t at
t
Thi
285 Enterobactergergoviae en p
s pa atpD
t
t
Thi
286 Enterobacter hormaechei en
s pa
287 Enterobactersakazakii This patent atpD
2gg Enterococcus avium This patent atpD
t atpD
t
Thi
289 Enterococcus casseliflavus en
s pa
290 Enterococcus durans This patent atpD
291 Enterococcus faecalis This patent atpD
2g2 Enterococcus faecium This patent atpD
293 Enterococcus gallinarum This patent atpD
294 Enterococcus saccharolyticus This patent atpD
295 Escherichia fergusonii This patent atpD
296 Escherichia hermannii This patent atpD
297 Escherichia vulneris This patent atpD
298 Eubacterium lentum This patent atpD
299 Ewingella americana This patent atpD
300 Francisella tularensis This patent atpD
301 Fusobacterium gonidiaformans This patent atpD
302 Fusobacterium necrophorum subsp. This patent atpD
necrophorum
303 Fusobacterium nucleatum subsp. polymorphumThis patent atpD
304 Gardnerella vaginalis This patent atpD
305 Gemella haemolysans This patent atpD
306 Gemella morbillorum This patent atpD
86
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEQ ID NO. Bacterial,fungal or parasitical species Source Comments*
307 Haemophilus ducreyi This patent afpD
308 Haemophilus haemolyticus This patent atpD
309 Haemophilus parahaemolyticus This patent atpD
310 Haemophilus parainfluenzae This patent atpD
311 Hafnia alvei This patent atpD
312 Kingella kingae This patent atpD
313 Klebsiella pneumoniae subsp. ozaenaeThis patent atpD
314 Klebsiella omithinolytica This patent atpD
315 Klebsiella oxytoca This patent atpD
316 Klebsiella planticola This patent atpD
317 Klebsiella pneumoniae subsp. pneumoniaeThis patent atpD
318 Kluyvera ascorbata This patent atpD
319 Kluyvera cryocrescens This patent atpD
t D
t at
Thi
320 Kluyvera georgiana en p
s pa atpD
t
t
Thi
321 Lactobacillus acidophilus en
s pa
322 Legionella pneumophila subsp. This patent atpD
pneumophila
323 Leminorella grimontii This patent atpD
324 Listeria monocytogenes This patent atpD
325 Micrococcus lylae This patent atpD
326 Moellerella wisconsensis This patent atpD
327 Moraxella catarrhalis This patent atpD
328 Moraxella osloensis This patent atpD
329 Morganella morganii subsp. morganiiThis patent atpD
330 Pantoea agglomerans This patent atpD
331 Pantoea dispersa This patent atpD
332 Pasteurella multocida This patent atpD
333 Pragia fontium This patent atpD
334 Proteus mirabilis This patent atpD
335 Proteus vulgaris This patent atpD
336 Providencia alcalifaciens This patent atpD
337 Providencia rettgeri This patent atpD
338 Providencia rustigianii This patent atpD
339 Providencia stuartii This patent atpD
340 Psychrobacter phenylpyruvicus This patent atpD
341 Rahnella aquatilis This patent atpD
342 Salmonella choleraesuis subsp. This patent atpD
arizonae
343 Salmonella choleraesuis subsp. This patent atpD
choleraesuis
serotype Choleraesuis
i This patent atpD
344 zonae t D
Salmonella choleraesuis subsp. t at
diar Thi
345 Salmonella choleraesuis subsp. en p
houtenae s pa
346 Salmonella choleraesuis subsp. This patent atpD
indica
347 Salmonella choleraesuis subsp. This patent atpD
choleraesuis
serotype Paratyphi A
choleraesuis This patent atpD
is subs
l
l
h
348 p.
eraesu
a c
o
Salmonel
serotype Paratyphi B
349 Salmonella choleraesuis subsp. This patent atpD
salamae
350 Salmonella choleraesuis subsp. atpD
choleraesuis serotype Typhi This
patent
351 Salmonella choleraesuis subsp. This patent atpD
choleraesuis
serotype Typhimurium
choleraesuis This patent atpD
esuis subs
l
h
ll
352 p.
era
o
a c
Salmone
serotype Virchow
This patent atpD
353 Serratia ficaria This patent atpD
354 Serratia fonticola This patent atpD
355 Serratia grimesii This patent atpD
356 Serratia liquefaciens This patent atpD
357 Serratia marcescens This patent atpD
358 Serratia odorifera This patent atpD
359 Serratia plymuthica This patent atpD
360 Serratia rubidaea This patent atpD
361 Pseudomonas putida This patent atpD
362 Shigella boydii This patent atpD
363 Shigella dysenteriae
87
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEGO ID NO. Bacterial,fungal or parasitical species Source Comments*
364 Shigella flexneri This patent atpD
365 Shigella sonnei This patent atpD
366 Staphylococcus aureus This patent atpD
367 Staphylococcus auricularis This patent atpD
368 Staphylococcus capitis subsp. This patent atpD
capitis
369 Staphylococcus cohnii This patent atpD
370 Staphylococcus epidermidis This patent atpD
371 Staphylococcus haemolyticus This patent atpD
372 Staphylococcus hominis subsp. This patent atpD
hominis
373 Staphylococcus hominis This patent atpD
374 Staphylococcus lugdunensis This patent atpD
375 Staphylococcus saprophyticus This patent atpD
376 Staphylococcus simulans This patent atpD
377 Staphylococcus warneri This patent atpD
t D
t at
hi
37g Streptococcus acidominimus en p
s pa atpD
T
t
t
Thi
379 Streptococcus agalactiae en at
s pa D
t
t
Thi
380 Streptococcus agalactiae en p
s pa at
t D
t
hi
381 Streptococcus agalactiae en p
s pa D
T at
t
t
Thi
382 Streptococcus agalactiae en p
s pa D
t at
t
Thi
383 Streptococcus agalactiae en p
s pa D
t at
t
hi
384 Streptococcus dysgalactiae en p
s pa at
T D
t
t
hi
385 Streptococcus equi subsp. equi en p
s pa
T
386 Streptococcus anginosus This patent atpD
t D
t at
Thi
387 Streptococcus salivarius en p
s pa D
t at
t
Thi
3gg Streptococcus suis en p
s pa D
t at
t
Thi
389 Streptococcus uberis en p
s pa D
t at
t
Thi
390 Tatumella ptyseos en p
s pa atpD
t
t
Thi
391 Trabulsiella guamensis en D
s pa at
t
t
hi
392 Yersinia bercovieri en p
s pa
T
393 Yersinia enterocolitica This patent atpD
394 Yersinia frederiksenii This patent atpD
395 Yersinia intermedia This patent atpD
396 Yersinia pseudotuberculosis This patent atpD
397 Yersinia rohdei This patent atpD
3gg Yokenella regensburgei This patent atpD
399 Yarrowia lipolytica This patent tuf (EF-1
)
400 Absidia corymbifera This patent tuf (EF-1
)
401 Alternaria alternata This patent tuf (EF-1
)
402 Aspergillus flavus This patent tuf (EF-1
)
403 Aspergillus fumigatus This patent tuf (EF-1
)
404 Aspergillus fumigatus This patent tuf (EF-1
)
405 Aspergillus niger This patent tuf (EF-1
)
406 Blastoschizomyces capitatus This patent tuf (EF-1
)
407 Candida albicans This patent tuf (EF-1
)
408 Candida albicans This patent tuf (EF-1
)
409 Candida albicans This patent tuf (EF-1
)
410 Candida albicans This patent tuf (EF-1
)
411 Candida albicans This patent tuf (EF-1
)
412 Candida dubliniensis This patent tuf (EF-1
)
413 Candida catenulata This patent tuf (EF-1
)
414 Candida dubliniensis This patent tuf(EF-1)
415 Candida dubliniensis This patent tuf (EF-1
)
416 Candida famata This patent tuf (EF-1
)
417 Candida glabrata This patent tuf (EF-1
)
418 Candida guilliermondii This patent tuf (EF-1
)
419 Candida haemulonii This patent tuf (EF-1
)
420 Candida inconspicua This patent tuf (EF-1
)
421 Candida kefyr This patent tuf (EF-1
)
422 Candida krusei This patent tuf (EF-1
)
423 Candida lambica This patent tuf (EF-1
)
424 Candida lusitaniae This patent tuf (EF-1
)
425 Candida norvegensis This patent tuf (EF-1
)
88
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEO ID NO. Bacterial,fungal or parasitical species Source Comments*
426 Candida parapsilosis This patent tuf (EF-1
)
427 Candida rugosa This patent tuf (EF-1
)
428 Candida sphaerica This patent tuf (EF-1
)
429 Candida tropicalis This patent tuf (EF-1
)
430 Candida utilis This patent tuf (EF-1
)
431 Candida viswanathii This patent tuf (EF-1
)
432 Candida zeylanoides This patent tuf (EF-1
)
433 Coccidioides immitis This patent tuf (EF-1
)
434 Cryptococcus albidus This patent tuf (EF-1
)
435 Exophiala jeanselmei This patent tuf (EF-1
)
436 Fusarium oxysporum This patent tuf (EF-1
t )
t tuf (EF-1
Thi )
437 Geotrichum sp. en tuf (EF-1
s pa )
This patent
438 Histoplasma capsulatum This patent tuf (EF-1
)
439 Issatchenkia orientalis kudrjanzevThis patent tuf (EF-1
)
440 Malassezia furfur This patent tuf (EF-1
)
441 Malassezia pachydermatis This patent tuf (EF-1
)
442 Malbranchea filamentosa This patent tuf (EF-1
)
443 Metschnikowia pulcherrima This patent tuf (EF-1
)
444 Paecilomyces lilacinus This patent tuf (EF-1
)
445 Paracoccidioides brasiliensis This patent tuf (EF-1
)
446 Penicillium marneffei This patent tuf (EF-1
)
447 Pichia anomala This patent tuf (EF-1
)
448 Pichia anomala This patent tuf (EF-1
)
449 Pseudallescheria boydii This patent tuf (EF-1
)
450 Rhizopus oryzae This patent tuf (EF-1
)
451 Rhodotorula minuta This patent tuf (EF-1
)
452 Sporobolomyces salmonicolor This patent tuf (EF-1
)
453 Sporothrix schenckii This patent tuf (EF-1
)
454 Stephanoascus ciferrii This patent tuf (EF-1
)
455 Trichophyton mentagrophytes This patent tuf (EF-1
)
456 Trichosporon cutaneum This patent tuf (EF-1
)
457 Wangiella dermatitidis This patent atpD
458 Aspergillus fumigatus This patent atpD
459 Blastoschizomyces capitatus This patent atpD
460 Candida albicans This patent atpD
461 Candida dubliniensis This patent atpD
462 Candida famata This patent atpD
463 Candida glabrata This patent atpD
464 Candida guilliermondii This patent atpD
465 Candida haemulonii This patent atpD
466 Candida inconspicua This patent atpD
467 Candida kefyr This patent atpD
468 Candida krusei This patent atpD
469 Candida lambica This patent atpD
470 Candida lusitaniae This patent atpD
471 Candida norvegensis This patent atpD
472 Candida parapsilosis This patent atpD
473 Candida rugosa This patent atpD
474 Candida sphaerica This patent atpD
475 Candida tropicalis This patent atpD
476 Candida utilis This patent atpD
477 Candida viswanathii This patent atpD
478 Candida zeylanoides This patent atpD
479 Coccidioides immitis This patent atpD
480 Cryptococcus albidus This patent atpD
481 Fusarium oxysporum This patent atpD
482 Geotrichum sp. This patent atpD
483 Histoplasma capsulatum This patent atpD
484 Malassezia furfur This patent atpD
485 Malassezia pachydermatis This patent atpD
486 Metschnikowia pulcherrima This patent atpD
487 Penicillium marneffei
89
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEQ ID NO. Bacterial,fungal or parasitical species Source Comments*
488 Pichia anomala This patent atpD
4gg Pichia anomala This patent atpD
490 Rhodotorula minuta This patent atpD
491 Rhodotorula mucilaginosa This patent atpD
492 Sporobolomyces salmonicolor This patent atpD
493 Sporothrix schenckii This patent atpD
494 Stephanoascus ciferrii This patent atpD
495 Trichophyton mentagrophytes This patent atpD
496 INangiella dermatitidis This patent atpD
4g7 Yarrowia lipolytica This patent atpD
498 Aspergillus fumigatus This patent tuf (M)
4gg Blastoschizomyces capitatus This patent tuf (M)
500 Candida rugosa This patent tuf (M)
501 Coccidioides immitis This patent tuf (M)
502 Fusarium oxysporum This patent tuf (M)
t tuf (M)
t
Thi
503 Histoplasma capsulatum en tuf (M)
s pa
t
t
i
504 Paracoccidioides brasiliensis en tuf (M)
s pa
Th
t
t
hi
505 Penicillium marneffei en tuf (M)
s pa
T
t
t
i
506 Pichia anomala en tuf (M)
s pa
Th
t
t
i
507 Trichophyton mentagrophytes en tuf (M)
s pa
Th
t
t
hi
508 Yarrowia lipolytica en tuf (EF-1
s pa )
T
t
t
Thi
509 Babesia bigemina en tuf (EF-1
s pa )
This patent
510 Babesia bovis This patent tuf (EF-1
)
511 Crithidia fasciculata This patent tuf (EF-1
)
512 Entamoeba histolytica This patent tuf (EF-1
)
513 Giardia lamblia This patent tuf (EF-1
)
514 Leishmania tropica This patent tuf (EF-1
)
515 Leishmania aethiopica This patent tuf (EF-1
)
516 Leishmania tropica This patent tuf (EF-1
)
517 Leishmania donovani This patent tuf(EF-1)
518 Leishmania infantum This patent tuf (EF-1
)
519 Leishmania enriettii This patent tuf (EF-1
)
520 Leishmania gerbilli This patent tuf (EF-1
)
521 Leishmania hertigi This patent tuf (EF-1
)
522 Leishmania major This patent tuf (EF-1
i )
523 s This patent tuf (EF-1
Leishmania amazonens )
524 Leishmania mexicana This patent tuf (EF-1
)
525 Leishmania tarentolae This patent tuf (EF-1
)
526 Leishmania tropica This patent tuf (EF-1
)
527 Neospora caninum This patent tuf (EF-1
)
528 Trichomonas vaginalis This patent tuf (EF-1
)
529 Trypanosoma brucei subsp. brucei t D
t at
Thi
530 Crithidia fasciculata en p
s pa
531 Leishmania tropica This patent atpD
532 Leishmania aethiopica This patent atpD
533 Leishmania donovani This patent atpD
534 Leishmania infantum This patent atpD
535 Leishmania gerbilli This patent atpD
536 Leishmania hertigi This patent atpD
537 Leishmania major This patent atpD
538 Leishmania amazonensis This patent atpD
607 Enterococcus faecalis W098/20157 tuf
608 Enterococcus faecium W098/20157 tuf
609 Enterococcus gallinarum W098/20157 tuf
610 Haemophilus influenzae Database tuf
611 Staphylococcus epidermidis W098/20157 tuf
612 Salmonella choleraesuis subsp. This patent tuf
choleraesuis
serotype Paratyphi A
This patent tuf
613 Serratia ficaria This patent tuf (C)
614 Enterococcus malodoratus This patent tuf (C)
615 Enterococcus durans This patent tuf (C)
616 Enterococcus pseudoavium
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SECT ID NO. Bacterial,fungal or parasitical species Source Comments*
617 Enterococcus dispar This patent tuf (C)
618 Enterococcus avium This patent tuf (C)
b tuf (M)
619 Saccharomyces cerevisiae ase tuf (C)
Data
t
t
Thi
621 Enterococcus faecium en tuf (EF-1
s pa )
t
t
i
622 Saccharomyces cerevisiae en tuf (EF-1
s pa )
Th
This patent
623 Cryptococcus neoformans This patent tuf (EF-1
)
624 Candida albicans W098/20157 tuf
662 Corynebacterium diphtheriae t atpD
t
Thi
663 Candida catenulata en tuf (EF-1
s pa )
t
b
D
665 Saccharomyces cerevisiae ase atpD
a
a
Database
666 Saccharomyces cerevisiae This patent atpD
667 Trypanosoma cruzi Database tuf
668 Corynebacterium glutamicum Database atpD
669 Escherichia colt Database atpD
670 Helicobacter pylori Database atpD
671 Clostridium acetobutylicum Database atpD
672 Cytophaga lytica This patent atpD
673 Ehrlichia risticii This patent atpD
674 Vibrio cholerae This patent tuf
675 Vibrio cholerae This patent atpD
676 Leishmania enriettii This patent tuf (EF-1
)
677 Babesia microti This patent atpD
678 Cryptococcus neoformans t at
t D
hi
67g Cryptococcus neoformans en p
s pa
T
680 Cunninghamella bertholletiae This patent atpD
684 Candida tropicalis Database atpD (V)
685 Enterococcus hirae Database atpD (V)
686 Chlamydia pneumoniae Database atpD (V)
687 Halobacterium salinarum Database atpD (V)
Egg Human Database atpD (V)
689 Plasmodium falciparum Database atpD (V)
690 Saccharomyces cerevisiae Database atpD (V)
b D (V)
at
691 Schizosaccharomyces pombe ase p
Data
692 Trypanosoma congolense Database atpD (V)
693 Thermus thermophilus Database atpD (V)
698 Escherichia colt Database tuf
7pg Borrelia burgdorferi genome project atpD (V)
710 Treponema pallidum genome project atpD (V)
711 Chlamydia trachomatis genome project atpD (V)
712 Enterococcus faecalis . genome project atpD (V)
713 Methanosarcina barkeri Database atpD (V)
714 Methanococcus jannaschii Database atpD (V)
715 Porphyromonas gingivalis genome project atpD (V)
716 Streptococcus pneumoniae genome project atpD (V)
717 Burkholderia mallet This patent tuf
718 Burkholderia pseudomallei This patent tuf
719 Clostridium beijerinckii This patent tuf
720 Clostridium innocuum This patent tuf
721 Clostridium novyi This patent tuf
722 Clostridium septicum This patent tuf
723 Clostridium tertium This patent tuf
724 Clostridium tetani This patent tuf
725 Enterococcus malodoratus This patent tuf
726 Enterococcus sulfureus This patent tuf
727 Lactococcus garvieae This patent tuf
f
t
728 Mycoplasma pirum This patent u
t
f
729 Mycoplasma salivarium This patent u
730 Neisseria polysaccharea This patent tuf
731 Salmonella choleraesuis subsp. This patent tuf
choleraesuis
serotype Enteritidis
91
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEO ID NO. Bacterial,fungal or parasitical species Source Comments*
$ 732 Salmonella choleraesuis subsp. This patent tuf
choleraesuis
serotype Gallinarum
choleraesuis This patent tuf
uis subs
l
l
h
733 p.
eraes
a c
o
Salmonel
serotype Paratyphi B
tuf
734 Salmonella choleraesuis subsp. This patent
choleraesuis
serotype Virchow
This patent tuf
735 Serratia grimesii This patent tuf
736 Clostridium difficile This patent atpD
737 Burkholderia pseudomallei t at
t D
hi
738 Clostridium bifermentans en p
s pa D
T at
t
t
Thi
739 Clostridium beijerinckii en p
s pa
740 Clostridium difficile This patent atpD
741 Clostridium ramosum This patent atpD
742 Clostridium septicum This patent atpD
743 Clostridium tertium This patent atpD
744 Comamonas acidovorans This patent atpD
745 Klebsiella pneumoniae subsp. rhinoscleromatisThis patent atpD
746 Neisseria cants This patent atpD
747 Neisseria cinerea This patent atpD
748 Neisseria cuniculi This patent atpD
749 Neisseria elongata subsp. elongataThis patent atpD
750 Neisseria flavescens This patent atpD
751 Neisseria gonorrhoeae This patent atpD
752 Neisseria gonorrhoeae This patent atpD
753 Neisseria lactamica This patent atpD
754 Neisseria meningitidis This patent atpD
755 Neisseria mucosa This patent atpD
756 Neisseria subflava This patent atpD
757 Neisseria weaveri This patent atpD
75g Neisseria animalis This patent atpD
759 Proteus penneri This patent atpD
760 Salmonella choleraesuis subsp. This patent atpD
choleraesuis
serotype Enteritidis
This patent atpD
761 Yersinia pestis This patent atpD
762 8urkholderia mallet This patent atpD
763 Clostridium sordellii This patent atpD
764 Clostridium novyi This patent atpD
765 Clostridium botulinum This patent atpD
766 Clostridium histolyticum This patent atpD
767 Peptostreptococcus prevotii t D
t at
Thi
768 Absidia corymbifera en p
s pa
769 Alternaria alternata This patent atpD
770 Aspergillus flavus This patent atpD
771 Mucor circinelloides This patent atpD
772 Piedraia hortai This patent atpD
773 Pseudallescheria boydii This patent atpD
774 Rhizopus oryzae This patent atpD
775 Scopulariopsis koningii This patent atpD
776 Trichophyton mentagrophytes This patent atpD
777 Trichophyton tonsurans This patent atpD
778 Trichosporon cutaneum This patent atpD
t tuf (EF-1
t )
Thi
779 Cladophialophora carrionii en tuf (EF-1
s pa )
t
t
Thi
780 Cunninghamella bertholletiae en
s pa
781 Curvularia lunata This patent tuf (EF-1
)
7g2 Fonsecaea pedrosoi This patent tuf (EF-1
)
783 Microsporum audouinii This patent tuf (EF-1
)
784 Mucor circinelloides This patent tuf (EF-1
)
785 Phialophora verrucosa This patent tuf (EF-1
)
786 Saksenaea vasiformis This patent tuf (EF-1
)
787 Syncephalastrum racemosum This patent tuf (EF-1
)
788 Trichophyton tonsurans This patent tuf (EF-1
t )
t tuf (EF-1
Thi )
789 Trichophyton mentagrophytes en
s pa
92
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEQ ID NO. Bacterial,fungal or parasitical species Source Comments*
790 Bipolaris hawaiiensis This patent tuf (EF-1
)
791 Aspergillus fumigatus This patent tuf (M)
792 Trichophyton mentagrophytes This patent tuf (M)
827 Clostridium novyi This patent atpD
(V)
828 Clostridium difficile This patent atpD
(V)
829 Clostridium septicum This patent atpD
(V)
830 Clostridium botulinum This patent atpD
(V)
831 Clostridium perfringens This patent atpD
(V)
832 Clostridium tetani This patent atpD
(V)
833 Streptococcus pyogenes Database atpD
(V)
834 Babesia bovis This patent atpD
(V)
835 Cryptosporidium parvum This patent atpD
(V)
836 Leishmania infantum This patent atpD
(V)
837 Leishmania major This patent atpD
(V)
838 Leishmania tarentolae This patent atpD
(V)
839 Trypanosoma brucei This patent atpD
(V)
840 Trypanosoma cruzi This patent tuf (EF-1
)
841 Trypanosoma cruzi This patent tuf (EF-1
)
842 Trypanosoma cruzi This patent tuf (EF-1
)
843 Babesia bovis This patent tuf (M)
844 Leishmania aethiopica This patent tuf (M)
845 Leishmania amazonensis This patent tuf (M)
846 Leishmania donovani This patent tuf (M)
847 Leishmania infantum This patent tuf (M)
848 Leishmania enriettii This patent tuf (M)
849 Leishmania gerbilli This patent tuf (M)
850 Leishmania major This patent tuf (M)
851 Leishmania mexicana This patent tuf (M)
852 Leishmania tarentolae This patent tuf (M)
853 Trypanosoma cruzi This patent tuf (M)
854 Trypanosoma cruzi This patent tuf (M)
855 Trypanosoma cruzi This patent tuf (M)
856 Babesia bigemina This patent atpD
857 Babesia bovis This patent atpD
858 Babesia microti This patent atpD
859 Leishmania guyanensis This patent atpD
860 Leishmania mexicana This patent atpD
861 Leishmania tropica This patent atpD
862 Leishmania tropica This patent atpD
863 Bordetella pertussis Database tuf
864 Trypanosoma brucei Database tuf (EF-1
)
865 Cryptosporidium parvum This patent tuf (EF-1
)
866 Staphylococcus saprophyticus This patent atpD
867 Zoogloea ramigera This patent atpD
868 Staphylococcus saprophyticus This patent tuf
869 Enterococcus casseliflavus This patent tuf
870 Enterococcus casseliflavus This patent tuf
871 Enterococcus flavescens This patent tuf
872 Enterococcus gallinarum This patent tuf
873 Enterococcus gallinarum This patent tuf
874 Staphylococcus haemolyticus This patent tuf
875 Staphylococcus epidermidis This patent tuf
876 Staphylococcus epidermidis This patent tuf
877 Staphylococcus epidermidis This patent tuf
878 Staphylococcus epidermidis This patent fuf
879 Enterococcus gallinarum This patent tuf
880 Pseudomonas aeruginosa This patent tuf
881 Enterococcus casseliflavus This patent tuf
882 Enterococcus casseliflavus This patent tuf
883 Enterococcus faecalis This patent tuf
884 Enterococcus faecalis This patent tuf
885 Enterococcus faecium This patent tuf
93
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEO ID NO. Bacterial,fungal or parasitical species Source Comments*
886 Enterococcus faecium This patent tuf
887 Zoogloea ramigera This patent tuf
888 Enterococcus faecalis This patent tuf
889 Aspergillus fumigatus This patent atpD
890 Penicillium marneffei This patent atpD
891 Paecilomyces lilacinus This patent atpD
892 Penicillium marneffei This patent atpD
893 Sporothrix schenckii This patent atpD
894 Malbranchea filamentosa This patent atpD
895 Paecilomyces lilacinus This patent atpD
896 Aspergillus niger This patent atpD
897 Aspergillus fumigatus This patent tuf (EF-1
)
898 Penicillium marneffei This patent tuf (EF-1
)
899 Piedraia hortai This patent tuf (EF-1
)
900 Paecilomyces lilacinus This patent tuf (EF-1
)
901 Paracoccidioides brasiliensis This patent tuf (EF-1
)
902 Sporothrix schenckii This patent tuf (EF-1
)
903 Penicillium marneffei This patent tuf (EF-1
)
904 Curvularia lunata This patent tuf (M)
905 Aspergillus niger This patent tuf (M)
906 Bipolaris hawaiiensis This patent tuf (M)
907 Aspergillus flavus This patent tuf (M)
908 Alternaria alternata This patent tuf (M)
909 Penicillium marneffei This patent tut (M)
910 Penicillium marneffei This patent tuf (M)
918 Escherichia coli Database recA
929 Bacteroides fragilis This patent atpD (V)
930 Bacteroides distasonis This patent atpD (V)
931 Porphyromonas asaccharolytica This patent atpD (V)
932 Listeria monocytogenes This patent tuf
939 Saccharomyces cerevisiae Database recA (Rad51)
940 Saccharomyces cerevisiae Database recA (Dmc1)
941 Cryptococcus humicolus This patent atpD
942 Escherichia coli This patent atpD
943 Escherichia coli This patent atpD
944 Escherichia coli This patent atpD
945 Escherichia coli This patent atpD
946 Neisseria polysaccharea This patent atpD
947 Neisseria sicca This patent atpD
948 Streptococcus mitis This patent atpD
949 Streptococcus mitis This patent atpD
950 Streptococcus mitis This patent atpD
951 Streptococcus oralis This patent atpD
952 Streptococcus pneumoniae This patent atpD
953 Streptococcus pneumoniae This patent atpD
954 Streptococcus pneumoniae This patent atpD
955 Streptococcus pneumoniae This patent atpD
956 Babesia microti This patent atpD (V)
957 Entamoeba hisfolytica This patent atpD (V)
958 Fusobacterium nucleatum subsp. This patent atpD (V)
polymorphum
959 Leishmania aethiopica This patent atpD (V)
960 Leishmania tropica This patent atpD (V)
961 Leishmania guyanensis This patent atpD (V)
962 Leishmania donovani This patent atpD (V)
963 Leishmania heriigi This patent atpD (V)
964 Leishmania mexicana This patent atpD (V)
965 Leishmania tropica This patent atpD (V)
966 Peptostreptococcus anaerobius This patent atpD (V)
967 Bordetella pertussis This patent tuf
968 Bordetella pertussis This patent tuf
969 Enterococcus columbae This patent tuf
94
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEO ID NO. Bacterial,fungal or parasitical species Source Comments*
970 Enterococcus flavescens This patent tuf
971 Streptococcus pneumoniae This patent tuf
972 Escherichia coli This patent tuf
973 Escherichia coli This patent tut
974 Escherichia coli This patent tuf
975 Escherichia coli This patent tuf
976 Mycobacterium avium This patent tuf
977 Streptococcus pneumoniae This patent tuf
978 Mycobacterium gordonae This patent tuf
979 Streptococcus pneumoniae This patent tuf
980 Mycobacterium tuberculosis This patent tuf
981 Staphylococcus warneri This patent tuf
982 Streptococcus mitis This patent tuf
983 Streptococcus mitis This patent tuf
984 Streptococcus mitis This patent tuf
985 Streptococcus oralis This patent tuf
986 Streptococcus pneumoniae This patent tuf
987 Enterococcus hirae This patent tuf
(C)
988 Enterococcus mundtii This patent tuf
(C)
989 Enterococcus raffinosus This patent tuf
(C)
990 Bacillus anthracis This patent recA
991 Prevotella melaninogenica This patent recA
992 Enterococcus casseliflavus This patent tuf
993 Streptococcus pyogenes Database speA
1002 Streptococcus pyogenes genome project tuf
1003 Bacillus cereus This patent recA
1004 Streptococcus pneumoniae This patent pbpla
1005 Streptococcus pneumoniae This patent pbpla
1006 Streptococcus pneumoniae This patent pbpla
1007 Streptococcus pneumoniae This patent pbpla
1008 Streptococcus pneumoniae This patent pbpla
1009 Streptococcus pneumoniae This patent pbpla
1010 Streptococcus pneumoniae This patent pbpla
1011 Streptococcus pneumoniae This patent pbpla
1012 Streptococcus pneumoniae This patent pbpla
1013 Streptococcus pneumoniae This patent pbpla
1014 Streptococcus pneumoniae This patent pbpla
1015 Streptococcus pneumoniae This patent pbpla
1016 Streptococcus pneumoniae This patent pbpia
1017 Streptococcus pneumoniae This patent pbpla
1018 Streptococcus pneumoniae This patent pbpla
1019 Streptococcus pneumoniae This patent pbp2b
1020 Streptococcus pneumoniae This patent pbp2b
1021 Streptococcus pneumoniae This patent pbp2b
1022 Streptococcus pneumoniae This patent pbp2b
1023 Streptococcus pneumoniae This patent pbp2b
1024 Streptococcus pneumoniae This patent pbp2b
1025 Streptococcus pneumoniae This patent pbp2b
1026 Streptococcus pneumoniae This patent pbp2b
1027 Streptococcus pneumoniae This patent pbp2b
1028 Streptococcus pneumoniae This patent pbp2b
1029 Streptococcus pneumoniae This patent pbp2b
1030 Streptococcus pneumoniae This patent pbp2b
1031 Streptococcus pneumoniae This patent pbp2b
1032 Streptococcus pneumoniae This patent pbp2b
1033 Streptococcus pneumoniae This patent pbp2b
1034 Streptococcus pneumoniae This patent pbp2x
1035 Streptococcus pneumoniae This patent pbp2x
1036 Streptococcus pneumoniae This patent pbp2x
1037 Streptococcus pneumoniae This patent pbp2x
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEO ID NO. Bacterial,fungal or parasitical species Source Comments*
1038 Streptococcus pneumoniae This patent pbp2x
1039 Streptococcus pneumoniae This patent pbp2x
1040 Streptococcus pneumoniae This patent pbp2x
1041 Streptococcus pneumoniae This patent pbp2x
1042 Streptococcus pneumoniae This patent pbp2x
1043 Streptococcus pneumoniae This patent pbp2x
1044 Streptococcus pneumoniae This patent pbp2x
1045 Streptococcus pneumoniae This patent pbp2x
1046 Streptococcus pneumoniae This patent pbp2x
1047 Streptococcus pneumoniae This patent pbp2x
1048 Streptococcus pneumoniae This patent pbp2x
1049 Enterococcus faecium This patent vanA
1050 Enterococcus gallinarum This patent vanA
1051 Enterococcus faecium This patent vanA
1052 Enterococcus faecium This patent vanA
1053 Enterococcus faecium This patent vanA
1054 Enterococcus faecalis This patent vanA
1055 Enterococcus gallinarum This patent vanA
1056 Enterococcus faecium This patent vanA
1057 Enterococcus flavescens This patent vanA
1058 Enterococcus gallinarum This patent vanCl
1059 Enterococcus gallinarum This patent vanC1
1060 Enterococcus casseliflavus This patent vanC2
1061 Enterococcus casseliflavus This patent vanC2
1062 Enterococcus casseliflavus This patent vanC2
1063 Enterococcus casseliflavus This patent vanC2
1064 Enterococcus flavescens This patent vanC3
1065 Enterococcus flavescens This patent vanC3
1066 Enterococcus flavescens This patent vanC3
1067 Enterococcus faecium This patent vanXY
1068 Enterococcus faecium This patent vanXY
1069 Enterococcus faecium This patent vanXY
1070 Enterococcus faecalis This patent vanXY
1071 Enterococcus gallinarum This patent vanXY
1072 Enterococcus faecium This patent vanXY
1073 Enterococcus flavescens This patent vanXY
1074 Enterococcus faecium This patent vanXY
1075 Enterococcus gallinarum This patent vanXY
1076 Escherichia coli Database stxi
1077 Escherichia coli Database stx2
1093 Staphylococcus saprophyticus This patent unknown
1117 Enterococcus faecium Database van8
1138 Enterococcus gallinarum Database vanC1
1139 Enterococcus faecium Database vanA
1140 Enterococcus casseliflavus Database vanC2
1141 Enterococcus faecium Database vanHAXY
1169 Streptococcus pneumoniae Database pbpla
1172 Streptococcus pneumoniae Database pbp2b
1173 Streptococcus pneumoniae Database pbp2x
1178 Staphylococcus aureus Database mecA
1183 Streptococcus pneumoniae Database hexA
1184 Streptococcus pneumoniae This patent hexA
1185 Streptococcus pneumoniae This patent hexA
1186 Streptococcus pneumoniae This patent hexA
1187 Streptococcus pneumoniae This patent hexA
96
CA 02307010 2000-OS-19
Table 7. Origin of the sequences in the sequence listing (continued).
SEO ID NO. Bacterial,fungal or parasitical species Source Comments*
1188 Streptococcus oralis This patent hexA
1189 Streptococcus mitis This patent hexA
1190 Streptococcus mitis This patent hexA
1191 Streptococcus mitis This patent hexA
1198 Staphylococcus saprophyticus This patent unknown
1215 Streptococcus pyogenes Database PCP
1230 Escherichia coli Database tuf (EF-G)
1242 Enterococcus faecium Database ddl
1243 Enterococcus faecalis Database mtlF,
mtlD
1244 Staphylococcus aureus subsp. This patent unknown
aureus
tuf indicates tufsequences, tuf (C) indicates tuf sequences divergent from
main (usually A and B) copies of the elongation factor-Tu,
tuf (EF-1) indicates tuf sequences of the eukaryotic type (elongation factor
1a), tuf (M) indicates tuf sequences from organellar
(mostly mitochondrial) origin.
atpD indicates atpD sequences of the F-type, aipD (V) indicates atpD sequences
of the V-type.
recA indicates recA sequences, recA(Rad51) indicates rad51 sequences or
homologs and recA(Dmci) indicates dmcl sequences or
homologs.
speA indicates speA sequences.
hexA indicates hexA sequences.
mecA indicates mecA sequences.
vanA indicates vanA sequences, van8 indicates van8 sequences, vanCl indicates
vanCf sequences, vanC2 indicates vanC2
sequences, vanC3indicates vanC3sequences, vanXYindicates vanXYsequences,
vanHAXYindicates vanHAXYsequences.
pbp is indicates pbpla sequences, pbp2b indicates pbp2b sequences, pbp2x
indicates pbp2x sequences.
pcp indicates pcp sequences.
stx, indicates stx, sequences, stx2 indicates stx2 sequences.
unknown indicates an unknown gene.
97
CA 02307010 2000-OS-19
Table 8. Bacterial species used to test the specificity of the Streptococcus
agalactiae-specific
amplification primers derived from tuf sequences.
Strain Reference number Strain Reference number
Streptococcus acidominimusATCC 51726 Bacteroides caccae ATCC 43185
Streptococcus agalactiaeATCC 12403 Bacteroides vulgatus ATCC 8482
Streptococcus agalactiaeATCC 12973 Bacteroides fragilis ATCC 25285
Streptococcus agalactiaeATCC 13813 Candida albicans ATCC 11006
Streptococcus agalactiaeATCC 27591 Clostridium innoculum ATCC 14501
Streptococcus agalactiaeCDCs 1073 Clostridium ramosum ATCC 25582
Streptococcus anginosusATCC 27335 Lactobacillus casei ATCC 393
subsp. casei
Streptococcus anginosusATCC 33397 Clostridium septicum ATCC 12464
Streptococcus bovis ATCC 33317 Corynebacterium cervicisNCTC 10604
Streptococcus anginosusATCC 27823 Corynebacterium genitaliumATCC 33031
Streptococcus cricetus ATCC 19642 Corynebacterium urealyticumATCC 43042
Streptococcus cristatusATCC 51100 Enterococcus faecalis ATCC 29212
Streptococcus downei ATCC 33748 Enterococcus faecium ATCC 19434
Streptococcus dysgalactiaeATCC 43078 Eubacterium lentum ATCC 43055
Streptococcus equi subsp.ATCC 9528 Eubacterium nodutum ATCC 33099
equi
Streptococcus ferns ATCC 33477 Gardnerella vaginalis ATCC 14018
Streptococcus gordonii ATCC 10558 Lactobacillus acidophilusATCC 4356
Streptococcus macacae ATCC 35911 Lactobacillus crispatusATCC 33820
Streptococcus mitis ATCC 49456 Lactobacillus gasseri ATCC 33323
Streptococcus mutans ATCC 25175 Lactobacillus johnsoniiATCC 33200
Streptococcus oralis ATCC 35037 Lactococcus lactis subsp.ATCC 19435
lactis
Streptococcus parasanguinisATCC 15912 Lactococcus lactis subsp.ATCC 11454
lactis
Streptococcus parauberisDSM 6631 Listeria innocua ATCC 33090
Streptococcus pneumoniaeATCC 27336 Micrococcus luteus ATCC 9341
Streptococcus pyogenes ATCC 19615 Escherichia coli ATCC 25922
Streptococcus ratti ATCC 19645 Micrococcus lylae ATCC 27566
Streptococcus salivariusATCC 7073 Porphyromonas asaccharolyticaATCC 25260
Streptococcus sanguinisATCC 10556 Prevotella corporis ATCC 33547
Streptococcus sobrinus ATCC 27352 Prevotella melanogenicaATCC 25845
Streptococcus suis ATCC 43765 Staphylococcus aureus ATCC 13301
Streptococcus uberis ATCC 19436 Staphylococcus epidermidisATCC 14990
Streptococcus vestubularisATCC 49124 Staphylococcus saprophyticusATCC 15305
98
CA 02307010 2000-OS-19
Table 9. Bacterial species used to test the specificity of the Streptococcus
agalactiae-specific
amplification primers derived from atpD sequences.
Strain Reference Strain Reference
number number
Streptococcus acidominimusATCC 51726 Streptococcus gordoniiATCC 10558
Streptococcus agalactiaeATCC 12400 Streptococcus macacaeATCC 35911
Streptococcus agalactiaeATCC 12403 Streptococcus mitis ATCC 49456
Streptococcus agalactiaeATCC 12973 Streptococcus mutansATCC 25175
Streptococcus agalactiaeATCC 13813 Streptococcus oralisATCC 35037
Streptococcus agalactiaeATCC 27591 Streptococcus parasanguinisATCC 15912
Streptococcus agalactiaeCDCs-1073 Streptococcus parauberisDSM 6631
Streptococcus anginosusATCC 27335 Streptococcus pneumoniaeATCC 27336
Streptococcus anginosusATCC 27823 Streptococcus pyogenesATCC 19615
Streptococcus bovis ATCC 33317 Streptococcus ratti ATCC 19645
Streptococcus cricetusATCC 19642 Streptococcus salivariusATCC 7073
Streptococcus cristatusATCC 51100 Streptococcus sanguinisATCC 10556
Streptococcus downei ATCC 33748 Streptococcus sobrinusATCC 27352
Streptococcus dysgalactiaeATCC 43078 Streptococcus suis ATCC 43765
Streptococcus equi ATCC 9528 Streptococcus uberisATCC 19436
subsp. equi
Streptococcus ferus ATCC 33477 Streptococcus vestibularisATCC 49124
99
CA 02307010 2000-OS-19
Table 10. Bacterial species used to test the specificity of the
Enterococcugspecific amplification
primers derived from fuf sequences.
Strain Reference number Strain Reference number
Gram-positive species (n=74)
Abiotrophia adiacens ATCC 49176 Listeria innocua ATCC 33090
Abiotrophia defectiva ATCC 49175 Listeria ivanovii ATCC 19119
Bacillus cereus ATCC 14579 Listeria monocytogenes ATCC 15313
Bacillus subtilis ATCC 27370 Listeria seeligeri ATCC 35967
Bifidobacterium adolescentisATCC 27534 Micrococcus luteus ATCC 9341
Bifidobacterium breve ATCC 15700 Pediococcus acidilacti ATCC 33314
Bifidobacterium dentiumATCC 27534 Pediococcus pentosaceusATCC 33316
Bifidobacterium longumATCC 15707 Peptococcus niger ATCC 27731
Clostridium perfringensATCC 3124 Peptostreptococcus anaerobiusATCC 27337
Clostridium septicum ATCC 12464 Peptostreptococcus indolicusATCC 29247
Corynebacterium aquaticusATCC 14665 Peptostreptococcus microsATCC 33270
Corynebacterium ATCC 10700 Propionibacterium acnesATCC 6919
pseudodiphtheriticum Staphylococcus aureus ATCC 43300
Enterococcus avium ATCC 14025 Staphylococcus capitis ATCC 27840
Enterococcus casseliflavusATCC 25788 Staphylococcus epidermidisATCC 14990
Enterococcus cecorum ATCC 43199 Staphylococcus haemolyticusATCC 29970
Enterococcus columbae ATCC 51263 Staphylococcus hominis ATCC 27844
Enterococcus dispar ATCC 51266 Staphylococcus lugdunensisATCC 43809
Enterococcus durans ATCC 19432 Staphylococcus saprophyticusATCC 15305
Enterococcus faecalis ATCC 29212 Staphylococcus simulansATCC 27848
Enterococcus faecium ATCC 19434 Staphylococcus warneri ATCC 27836
Enterococcus flavescensATCC 49996 Streptococcus agalactiaeATCC 13813
Enterococcus gallinarumATCC 49573 Streptococcus anginosusATCC 33397
Enterococcus hirae ATCC 8044 Streptococcus bovis ATCC 33317
Enterococcus malodoratusATCC 43197 Streptococcus constellatusATCC 27823
Enterococcus mundtii ATCC 43186 Streptococcus cristatusATCC 51100
Enterococcus pseudoaviumATCC 49372 Streptococcus intermediusATCC 27335
Enterococcus raffinosusATCC 49427 Streptococcus mitis ATCC 49456
Enterococcus saccharolyticusATCC 43076 Streptococcus mitis ATCC 3639
Enterococcus solitariusATCC 49428 Streptococcus mutans ATCC 27175
Enterococcus sulfureusATCC 49903 Streptococcus parasanguinisATCC 15912
Eubacterium lentum ATCC 49903 Streptococcus pneumoniaeATCC 27736
Gemella haemolysans ATCC 10379 Streptococcus pneumoniaeATCC 6303
Gemella morbillorum ATCC 27842 Streptococcus pyogenes ATCC 19615
Lactobacillus acidophilusATCC 4356 Streptococcus salivariusATCC 7073
Leuconostoc mesenteroidesATCC 19225 Streptococcus sanguinisATCC 10556
Listeria grayi ATCC 19120 Streptococcus suis ATCC 43765
Listeria grayi ATCC 19123
100
CA 02307010 2000-OS-19
Table 10. Bacterial species used to test the specificity of the Enterococcus
genus-specific
amplification primers derived from tuf sequences (continued).
Strain Reference number Strain Reference number
Gram-negative species (n=39)
Acidominococcus fermentansATCC 2508 Hafnia alvei ATCC 13337
Acinetobacter baumanniiATCC 19606 Klebsiella oxytoca ATCC 13182
Alcaligenes faecalis ATCC 8750 Meganomonas hypermegasATCC 25560
Anaerobiospirillum ATCC 29305 Mitsukoella multiacidusATCC 27723
succiniproducens Moraxella catarrhalis ATCC 43628
Anaerorhabdus furcosusATCC 25662 Morganella morganii ATCC 25830
Bacteroides distasonisATCC 8503 Neisseria meningitidisATCC 13077
Bacteroides thetaiotaomicronATCC 29741 Pasteurella aerogenes ATCC 27883
Bacteroides vulgatus ATCC 8482 Proteus vulgaris ATCC 13315
Bordetella pertussis LSPQ 3702 Providencia alcalifaciensATCC 9886
Bulkholderia cepacia LSPQ 2217 Providencia rettgeri ATCC 9250
Butyvibrio fibrinosolvensATCC 19171 Pseudomonas aeruginosaATCC 27853
Cardiobacterium hominisATCC 15826 Salmonella typhimuriumATCC 14028
Citrobacter freundii ATCC 8090 Serratia marcescens ATCC 13880
Desulfovibrio vulgarisATCC 29579 Shigella flexneri ATCC 12022
Edwardsiellae tarda ATCC 15947 Shigella sonnei ATCC 29930
Enterobacter cloacae ATCC 13047 Succinivibrio dextrinosolvensATCC 19716
Escherichia coli ATCC 25922 Tissierella praeacuta ATCC 25539
Fusobacterium russii ATCC 25533 Veillonella parvula ATCC 10790
Haemophilus influenzaeATCC 9007 Yersinia enterocoliticaATCC 9610
101
CA 02307010 2000-OS-19
Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases.
Species Strain Accession number Coding gene*
Bacteria
Agrobacterium tumefaciens X99673 tuf
Agrobacterium tumefaciens X99673 tuf (EF-G)
Agrobacterium tumefaciens X99674 tuf
Anacystis nidulans PCC 6301 X17442 tuf
Aquifex aeolicus VF5 AE000669 tuf
Aquifex aeolicus VF5 AE000669 tuf (EF-G)
Aquifex pyrophilus Genome project2tuf (EF-G)
Aquifex pyrophilus Y15787 tuf
Archaeoglobus fulgidus Genome project2tuf (EF-G)
Bacillus halodurans C-125 AB017508 tuf
Bacillus halodurans C-125 AB017508 tuf (EF-G)
Bacillus stearothermophilusCCM 2184 AJ000260 tuf
Bacillus subtilis 168 D64127 tuf
Bacillus subtilis 168 D64127 tuf (EF-G)
Bacillus subtilis DSM 10 299104 tuf
Bacteroides forsythus ATCC 43037 AB035466 tuf
Bacteroides fragilis DSM 1151 -' tuf
Bordetella bronchisepticaRB50 Genome project2tuf
Bordetella pertussis Tohama 1 Genome project2tuf
Borrelia burdorgferi B31 078193 tuf
Borrelia burgdorferi AE001155 tuf (EF-G)
Brevibacterium linens DSM 20425 X76863 tuf
Buchnera aphidicola Ap Y12307 tuf
Campylobacter jejuni NCTC 11168 Y17167 tuf
Chlamydia pneumoniae CWL029 AE001592 tuf
Chlamydia pneumoniae CWL029 AE001639 tuf (EF-G)
Chlamydia trachomatis M74221 tuf
Chlamydia trachomatis D/UW-3/CX AE001317 tuf (EF-G)
Chlamydia trachomatis D/UW-3/CX AE001305 tuf
Chlamydia trachomatis F/IC-Cal-13 L22216 tuf
Chlorobium vibrioforme DSM 263 X77033 tuf
Chloroflexus aurantiacusDSM 636 X76865 tuf
Clostridium acetobutylicumATCC 824 Genome project2tuf
Clostridium difficile 630 Genome project2tuf
Corynebacterium glutamicumASO 19 X77034 tuf
Corynebacterium glutamicumMJ-233 E09634 tuf
Coxiella burnetii Nine Mile phase AF136604 tuf
I
Cytophaga lytica DSM 2039 X77035 tuf
Deinococcus radioduransR1 AE001891 tuf (EF-G)
Deinococcus radioduransR1 Genome project2tuf
Deinonema sp. -' tuf
Eikenella corrodens ATCC 23834 212610 tuf
Eikenella corrodens ATCC 23834 212610 tuf (EF-G)
Enterococcus faecalis Genome project2tuf (EF-G)
Escherichia coli J01690 tuf
Escherichia coli J01717 tuf
Escherichia coli X00415 tuf (EF-G)
Escherichia coli X57091 tuf
Escherichia coli K-12 MG1655 000006 tuf
Escherichia coli K-12 MG1655 000096 tuf
Fervidobacterium islandicumDSM 5733 Y15788 tuf
Fibrobacter succinogenesS85 X76866 tuf
102
CA 02307010 2000-OS-19
Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
Flavobacterium ferrigeneumDSM 13524 X76867 tuf
Flexistipes sinusarabici X59461 tuf
Gloeobacter violaceus PCC 7421 009433 tuf
Gloeothece sp. PCC 6501 009434 tuf
Haemophilus actinomycetemcomitansHK1651 Genome project2 tuf
Haemophilus ducreyi 35000 AF087414 tuf (EF-G)
Haemophilus influenzae Rd 032739 tuf
Haemophilus influenzae Rd 032739 tuf (EF-G)
Halobacterium marismortui X16677 tuf
Helicobacter pylori 26695 AE000511 tuf
Helicobacter pylori J99 AE001539 tuf (EF-G)
Helicobacter pylori J99 AE001541 tuf
Herpetosiphon aurantiacus Hpga1 X76868 tuf
Lactobacillus paracasei E13922 tuf
Leptospira interrogans AF115283 tuf
Methanobacterium thermoautrophicumdelta H AE000877 tuf
Methanococcus jannaschii ATCC 43067 067486 tuf
Methanococcus jannaschii DSM 2661 Genome project2 tuf (EF-G)
Methanococcus vannielii X05698 fuf
Micrococcus luteus IFO 3333 M17788 tuf(EF-G)
Micrococcus luteus IFO 3333 M17788 tuf
Moraxella sp. TAC I I 25 AJ249258 tuf
Mycobacterium avium 104 Genome project2 tuf
Mycobacterium bovis AF2122/97 Genome project2 tuf
Mycobacterium leprae L13276 tuf
Mycobacterium leprae 214314 tuf
Mycobacterium leprae 214314 tuf (EF-G)
Mycobacterium leprae Thai 53 D13869 tuf
Mycobacterium tuberculosisErdmann S40925 tuf
Mycobacterium tuberculosisH37Rv AL021943 tuf (EF-G)
Mycobacterium tuberculosisH37Rv 284395 tuf
Mycobacterium tuberculosisy42 AD000005 tuf
Mycoplasma capricolum PG-31 X16462 tuf
Mycoplasma genitalium G37 039732 tuf
Mycoplasma genitalium G37 Genome project2 tuf (EF-G)
Mycoplasma hominis X57136 tuf
Mycoplasma hominis PG21 M57675 tuf
Mycoplasma pneumoniae M129 AE000019 tuf
Mycoplasma pneumoniae M129 AE000058 tuf(EF-G)
Neisseria gonorrhoeae MS11 L36380 tuf
Neisseria gonorrhoeae MS11 L36380 tuf (EF-G)
Neisseria meningitidis Genome project2 tuf (EF-G)
Neisseria meningitidis 22491 Genome project2 tuf
Peptococcus niger DSM 20745 X76869 tuf
Porphyromonas gingivalis W83 Genome project2 tuf
Phormidium ectocarpi PCC 7375 009443 tuf
Planobispora roses ATCC 53773 067308 tuf
Plectonema boryanum PCC 73110 009444 tuf
Porphyromonas gingivalis W83 Genome project2 tuf
Porphyromonas gingivalis FDC 381 AB035461 tuf
Porphyromonas gingivalis W83 AB035462 tuf
Porphyromonas gingivalis SUNY 1021 AB035463 tuf
Porphyromonas gingivalis A7A1-28 AB035464 tuf
Porphyromonas gingivalis ATCC 33277 AB035465 tuf
Porphyromonas gingivalis ATCC 33277 AB035471 tuf (EF-G)
103
CA 02307010 2000-OS-19
Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
Prochlorothrix hollandica U09445 tuf
Pseudomonas aeruginosa PAO-1 Genome projectztuf
Pyrococcus abyssi Orsay AJ248285 tuf
Rickettsia prowazekii Madrid E AJ235272 tuf
Rickettsia prowazekii Madrid E 254171 tuf (EF-G)
Salmonella typhimurium X64591 tuf (EF-G)
Salmonella typhimurium LT2 trpE91 X55116 tuf
Salmonella typhimurium LT2 trpE91 X55117 tuf
Serpulina hyodysenteriae8204 U51635 tuf
Serratia marcescens AF058451 tuf
Shewanella putida Genome project2tuf
Shewanella putrefaciensDSM 50426 -' tuf
Shewanella putrefaciensMR-1 Genome project2tuf
Spirochaeta aurantia DSM 1902 X76874 tuf
Staphylococcus aureus AJ237696 tuf (EF-G)
Stigmatella aurantiaca DW4 X82820 tuf
Stigmatella aurantiaca Sg a1 X76870 tuf
Streptococcus mutans GS-5 Kuramitsu U75481 tuf
Streptococcus mutans UAB159 Genome project2tuf
Streptococcus oralis NTCC 11427 P331701 tuf
Streptococcus pyogenes Genome project2tuf (EF-G)
Streptococcus pyogenes M1-GAS Genome project2tuf
Streptomyces aureofaciensATCC 10762 AF007125 tuf
Streptomyces cinnamoneusTue89 X98831 tuf
Streptomyces coelicolorA3(2) AL031013 tuf (EF-G)
Streptomyces coelicolorM 145 X77039 tuf
Streptomyces collinus BSM 40733 S79408 tuf
Streptomyces netropsis Tu1063 AF153618 tuf
Streptomyces ramocissimus X67057 tuf
Streptomyces ramocissimus X67057 tuf (EF-G)
Synechococcus sp. X17442 tuf (EF-G)
Synechocystis sp. X65159 tuf (EF-G)
Synechocystis sp. PCC 6803 D90913 tuf
Taxeobacter occealus Myx 2105 X77036 tuf
Thermoplasma acidophilumDSM 1728 X53866 tuf
Thermotoga maritima Genome project2tuf (EF-G)
Thermotoga maritima M27479 tuf
Thermos aquaticus EP 00276 X66322 tuf
Thermos thermophilus HB8 X16278 tuf (EF-G)
Thermos thermophilus HB8 X05977 tuf
Thermos thermophilus HB8 X06657 tuf
Thiomonas cuprina DSM 5495 U78300 tuf
Thiomonas cuprina DSM 5495 U78300 tuf (EF-G)
Thiomonas cuprina Hoes X76871 tuf
Treponema pallidum AE001202 tuf
Treponema pallidum AE001248 tuf (EF-G)
Streptomyces ramocissimus X67058 tuf
Ureaplasma urealyticum ATCC 33697 234275 tuf
Vibrio cholerae N16961 TIGR2 tuf
Wolinella succinogenes DSM 1740 X76872 tuf
Yersinia pesos CO-92 Genome projectztuf
Fungi
Absidia glauca CBS 101.48 X54730 tuf (EF-1 )
104
CA 02307010 2000-OS-19
Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
Arxula adeninivorans Ls3 247379 tuf (EF-1
)
Aspergillus oryzae KBN616 AB007770 tuf (EF-1
)
Aureobasidium pullulans8106 U19723 tuf (EF-1
)
Candida albicans SC5314 M29934 tuf (EF-1
)
Candida albicans SC5314 M29935 tuf (EF-1
)
Cryptococcus neoformans83501 U81803 tuf (EF-1
)
Cryptococcus neoformansM1-106 U81804 tuf (EF-1
)
Eremothecium gossypii ATCC 10895 X73978 tuf (EF-1
)
Fusarium oxysporum NRRL 26037 AF008498 tuf (EF-1
)
Histoplasma capsulatum 186AS U14100 tuf (EF-1
)
Podospora anserina X74799 tuf (EF-1
)
Puccinia graminis race 32 X73529 tuf (EF-1
)
Rhizomucor racemosus ATCC 12168 X17475 tuf (EF-1
)
Rhizomucor racemosus ATCC 12168 J02605 tuf (EF-1
)
Rhizomucor racemosus ATCC 1216B X17476 tuf (EF-1
)
Rhodotorula mucilaginosa AF016239 tuf (EF-1
)
Saccharomyces cerevisiae K00428 tuf (M)
Saccharomyces cerevisiae M59369 tuf (EF-G)
Saccharomyces cerevisiae X00779 tuf (EF-1
)
Saccharomyces cerevisiae X01638 tuf (EF-1
)
Schizosaccharomyces U42189 tuf (EF-1
pombe )
Trichoderma reesei OM9414 223012 tuf (EF-1
)
Yarrowia lipolytica AF054510 tuf (EF-1
)
Parasites
Blastocystis hominis HE87-1 D64080 tuf (EF-1
)
Eimeria tenella LS18 AI755521 tuf (EF-1
)
Giardia lamblia D14342 tuf (EF-1
)
Kentrophoros sp. AF056101 tuf (EF-1
)
Leishmania amazonensisIFLA/BR/67/PH8 M92653 tuf (EF-1
)
Leishmania braziliensis U72244 tuf (EF-1
)
Onchocerca volvulus M64333 tuf (EF-1
)
Porphyra purpurea Avonport U08844 tuf (EF-1
)
Plasmodium berghei ANKA AJ224150 tuf (EF-1
)
Plasmodium falciparum K1 X60488 tuf (EF-1
)
Plasmodium knowlesi line H AJ224153 tuf (EF-1
)
Toxoplasma gondii RH Y11431 tuf (EF-1
)
Trypanosoma cruzi Y L76077 tuf (EF-1
)
Trypanosoma brucei LVH/75/ U10562 tuf (EF-1
)
USAMRU-K/18
Human and plants
Arabidopsis thaliana Columbia X89227 tuf (EF-1
)
Glycine max Ceresia X89058 tuf (EF-1
)
Glycine max Ceresia Y15107 tuf (EF-1
)
Glycine max Ceresia Y15108 tuf (EF-1
)
Glycine max Maple Arrow X66062 tuf (EF-1
)
Homo sapiens X03558 tuf (EF-1
)
Pyramimonas disomata AB008010 tuf
Bacteria
Acetobacterium woodi DSM 1030 U10505 atpD
Bacillus firmus OF4 M60117 atpD
105
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Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
Bacillus megaterium OM 81551 M20255 atpD
Bacillus stearothermophilus D38058 atpD
Bacillus stearothermophilusIF01035 D38060 atpD
Bacillus subtilis 168 228592 atpD
Bacteroides fragilis DSM 2151 M22247 atpD
Bordetella bronchisepticaRB50 Genome project2 atpD
Bordetella pertussis Tohama 1 Genome project2 atpD
Borrelia burgdorferi 831 AE001122 atpD
(V)
Burkholderia cepacia DSM50181 X76877 atpD
Campylobacter jejuni NCTC 11168 Genome project2 atpD
Chlamydia pneumoniae Genome project2 atpD
(V)
Chlamydia trachomatis MoPn Genome project2 atpD
(V)
Chlorobium vibrioforme DSM 263 X76873 atpD
Citrobacter freundii JE0503 AF037156 atpD
Clostridium acetobutylicumATCC 824 Genome project2 atpD
Clostridium acetobutylicumDSM 792 AF101055 atpD
Clostridium difficile 630 Genome project2 atpD
Corynebacterium glutamicumASO 19 X76875 atpD
Corynebacterium glutamicumMJ-233 E09634 atpD
Cytophaga lytica DSM 2039 M22535 atpD
Enterobacter aerogenes DSM 30053 -3 atpD
Enterococcus faecalis M90060 atpD
Enterococcus faecalis V583 Genome project2 atpD
(V)
Enterococcus hirae ATCC 9790 D17462 atpD
(V)
Escherichia coli J01594 atpD
Escherichia coli M25464 atpD
Escherichia coli V00267 atpD
Escherichia coli V00311 atpD
Escherichia coli K12 MG1655 L10328 atpD
Flavobacterium ferrugineumDSM 13524 -3 atpD
Haemophilus actinomycetemcomitans Genome project2 atpD
Haemophilus influenzae Rd 032730 atpD
Halobacterium salinarum S56356 atpD
(V)
Haloferax volcanii WR 340 X79516 atpD
Helicobacter pylori NCTC 11638 AF004014 atpD
Helicobacter pylori 26695 Genome project2 atpD
Helicobacter pylori J99 Genome project2 atpD
Lactobacillus casei DSM 20021 X64542 atpD
Methanococcus jannaschii DSM 2661 067477 atpD
(V)
Methanosarcina barkeri DSM 800 J04836 atpD
(V)
Moorella thermoacetica ATCC 39073 064318 atpD
Mycobacterium avium 104 Genome project2 atpD
Mycobacterium bovis AF2122/97 Genome project2 atpD
Mycobacterium leprae 015186 atpD
Mycobacterium tuberculosisH37Rv 273419 atpD
Mycoplasma gallisepticum X64256 atpD
Mycoplasma genitalium G37 039725 atpD
Mycoplasma pneumoniae M129 043738 atpD
Neisseria gonorrhoeae FA 1090 Genome project2 atpD
Neisseria meningitidis 22491 Genome project2 atpD
Pectinatus frisingensis DSM 20465 X64543 atpD
Peptococcus niger DSM 20475 X76878 atpD
Pirellula marina IFAM 1313 X57204 atpD
Porphyromonas gingivalis W83 Genome project2 atpD
(V)
Propionigenium modestum DSM 2376 X58461 atpD
106
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Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
Pseudomonas aeruginosa PA01 Genome project2 atpD
2
Pseudomonas putida Genome project atpD
Rhodobacter capsulatus 8100 X99599 atpD
Rhodospirillum rubrum X02499 atpD
Rickettsia prowazekii F-12 AF036246 2 atpD
Rickettsia prowazekii Madrid Genome project atpD
Ruminococcus albus 7ATCC AB006151 atpD
Salmonella choleraesuis S83769 AF037146 atpD
subsp. arizonae
Salmonella choleraesuis u24 AF037147 atpD
subsp. arizonae
Salmonella bongori JE04162 AF037155 atpD
Salmonella bongori BR1859 AF037154 atpD
Salmonella choleraesuis DS210/89 AF037149 atpD
subsp. diarizonae
Salmonella choleraesuis JE0307 AF037148 atpD
subsp. diarizonae
Salmonella choleraesuis S109671 AF037150 atpD
subsp. diarizonae
Salmonella choleraesuis K228 AF037140 atpD
subsp.
choleraesuis serotype
Dublin 7139 D
at
Salmonella choleraesuis K771 AF03 p
subsp.
choleraesuis serotype
Dublin AF037151 D
at
Salmonella choleraesuis S84366 p
subsp. houtenae
Salmonella choleraesuis S84098 AF037152 atpD
subsp. houtenae
Salmonella choleraesuis BR2047 AF037153 atpD
subsp. indica
Salmonella choleraesuis Div36-86 AF037142 atpD
subsp.
choleraesuis serotype
Infantis AF037144 at
D
Salmonella choleraesuis NSC72 p
subsp. salamae
Salmonella choleraesuis S114655 AF037145 atpD
subsp. salamae
Salmonella choleraesuis Div95-86 AF037143 atpD
subsp.
choleraesuis serotype
Tennessee AF037141 at
D
Salmonella choleraesuis LT2 p
subsp.
choleraesuis serotype
Typhimurium 2
Shewanella putida Genome project atpD
2
Shewanella putrefaciens MR-1 Genome project atpD
Stigmatella aurantiaca Sga1 X76879 atpD
Streptococcus bovis JB-1 AB009314 atpD
Streptococcus mutans GS-5 U31170 atpD
2
Streptococcus mutans UAB159 Genome project atpD
Streptococcus pneumoniae Type 4 Genome project2 atpD (V)
Streptococcus pneumoniae Type 4 Genome project2 afpD
Streptococcus pyogenes Genome project atpD (V)
2
Streptococcus pyogenes M1-GAS Genome project atpD
Streptococcus sanguinis 10904 AF001955 atpD
Streptomyces lividans 1326 222606 atpD
Thermus thermophilus HB8 D63799 atpD (V)
Thiobacillus ferrooxidansATCC 33020 M81087 atpD
Treponema pallidum Nichols AE001228 atpD (V)
Vibrio alginolyticus X16050 atpD
107
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Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
Vibrio cholerae N16961 Genome project2 atpD
INolinella succinogenesDSM 1470 X76880 atpD
Yersinia enterocoliticaNCTC 10460 AF037157 atpD
2
Yersinia pestis CO-92 Genome project atpD
Fungi
Candida tropicalis M64984 atpD (V)
Kluyveromyces lactis 2359/152 U37764 atpD
Neurospora crassa X53720 atpD
Saccharomyces cerevisiae M12082 atpD
409 atpD (V)
Saccharomyces cerevisiae X2180-1 A J05 atpD (V)
S47814
Schizosaccharomyces pombe 972 h-
Schizosaccharomyces pombe 972 h- M57956 atpD
Parasites
Giardia lamblia WB U18938 atpD
Plasmodium falciparum 3D7 L08200 atpD (V)
Trypanosoma congolense IL3000 225814 atpD (V)
Human and plants
Homo sapiens L09234 atpD (V)
Homo sapiens M27132 atpD
Bacteria
Acetobacter aceti no. 1023 560630 recA
Acetobacter altoacetigenesMH-24 E05290 recA
Acetobacter polyoxogenesNBI 1028 D13183 recA
Acholeplasma laidlawii 8195 M81465 recA
Acidiphilium facilis ATCC 35904 D16538 recA
Acidothermus cellulolyticusATCC 43068 AJ006705 recA
Acinetobacter calcoaceticusBD413/ADP1 L26100 recA
Aeromonas salmonicida A449 U83688 recA
Agrobacterium tumefaciensC58 L07902 recA
Allochromatium vinosum AJ000677 recA
Aquifex aeolicus VF5 AE000775 recA
Aquifex pyrophilus KolSa L23135 recA
Azotobacter vinelandii S96898 recA
2
Bacillus stearothermophilus10 Genome project recA
Bacillus subtilis PB1831 U87792 recA
Bacillus subtilis 168 299112 recA
Bacteroides fragilis M63029 recA
Bifidobacterium breve NCFB 2258 AF094756 recA
Blastochloris viridis DSM 133 AF022175 recA
Bordetella pertussis 165 X53457 recA
Borrelia burgdorferi Sh-2-82 U23457 recA
Borrelia burgdorferi B31 AE001124 recA
Brevibacterium flavum MJ-233 E10390 recA
Brucella abortus 2308 L00679 recA
Burkholderia cepacia ATCC 17616 U70431 recA
Campylobacter jejuni 81-176 U03121 recA
Chlamydia trachomatis L2 U16739 recA
108
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Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
Chloroflexus aurantiacus J-10-fl AF037259 recA
Clostridium acetobutylicum M94057 recA
Clostridium perfringens 13 061497 recA
Corynebacterium glutamicumAS019 014965 recA
Corynebacterium pseudotuberculosisC231 030387 recA
Deinococcus radiodurans KD8301 AB005471 recA
Deinococcus radiodurans R1 001876 recA
Enterobacter agglomerans 339 L03291 recA
Enterococcus faecalis OGIX M81466 recA
Erwinia carotovora X55554 recA
Escherichia coli J01672 recA
Escherichia coli X55552 recA
Escherichia coli K-12 AE000354 recA
Frankia alni Arl3 AJ006707 recA
Gluconobacter oxydans 021001 recA
Haemophilus influenzae Rd 032687 recA
Haemophilus influenzae Rd 032741 recA
Haemophilus influenzae Rd L07529 recA
Helicobacter pylori 69A 235478 recA
Helicobacter pylori 26695 AE000536 recA
Helicobacter pylori J99 AE001453 recA
Lactococcus lactis ML3 M88106 recA
Legionella pneumophila X55453 recA
Leptospira biflexa serovar patoc032625 recA
Leptospira interrogans serovar pomona029169 recA
Magnetospirillum magnetotacticumMS-1 X17371 recA
Methylobacillus flagellatusMFK1 M35325 recA
Methylomonas clara ATCC 31226 X59514 recA
Mycobacterium leprae X73822 recA
Mycobacterium tuberculosisH37Rv X58485 recA
Mycoplasma genitalium G37 039717 recA
Mycoplasma mycoides GM9 L22073 recA
Mycoplasma pneumoniae ATCC 29342 MPAE000033 recA
Mycoplasma pulmonis KD735 L22074 recA
Myxococcus xanthus L40368 recA
Neisseria animalis NCTC 10212 057910 recA
Neisseria cinerea LCDC 81-176 AJ223869 recA
Neisseria cinerea LNP 1646 057906 recA
Neisseria cinerea NCTC 10294 AJ223871 recA
Neisseria cinerea Vedros M601 AJ223870 recA
Neisseria elongata CCUG 2131 AJ223882 recA
Neisseria elongata CCUG 4165A AJ223880 recA
Neisseria elongata CCUG 4557 AJ223879 recA
subsp. intermedia
Neisseria elongata NCTC 10660 AJ223881 recA
Neisseria elongata NCTC 11050 AJ223878 recA
Neisseria elongata NHITCC 2376 AJ223877 recA
Neisseria flava Bangor 9 AJ223873 recA
Neisseria flavescens LNP 444 057907 recA
Neisseria gonorrhoeae CH95 057902 recA
Neisseria gonorrhoeae FA19 X64842 recA
Neisseria gonorrhoeae MS11 X17374 recA
Neisseria lactamica CCUC 7757 AJ223866 recA
Neisseria lactamica CCUG 7852 Y11819 recA
Neisseria lactamica LCDC 77-143 Y11818 recA
109
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Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
Neisseria lactamica LCDC 80-111 AJ223864 recA
Neisseria lactamica LCDC 845 AJ223865 recA
Neisseria lactamica NCTC 10617 057905 recA
Neisseria lactamica NCTC 10618 AJ223863 recA
Neisseria meningitides 44/46 X64849 recA
Neisseria meningitides Bangor 13 AJ223868 recA
Neisseria meningitides HF116 X64848 recA
Neisseria meningitides HF130 X64844 recA
Neisseria meningitides HF46 X64847 recA
Neisseria meningitides M470 X64850 recA
Neisseria meningitides N9411 X64846 recA
Neisseria meningitides NCTC 8249 AJ223867 recA
Neisseria meningitides P63 X64845 recA
Neisseria meningitides S3446 057903 recA
Neisseria mucosa LNP 405 057908 recA
Neisseria mucosa Vedros M1801 AJ223875 recA
Neisseria perflava CCUG 17915 AJ223876 recA
Neisseria perflava LCDC 85402 AJ223862 recA
Neisseria pharynges var.NCTC 4590 057909 recA
flava
Neisseria polysaccharea CCUG 18031 Y11815 recA
Neisseria polysaccharea CCUG 24845 Y11816 recA
Neisseria polysaccharea CCUG 24846 Y11814 recA
Neisseria polysaccharea INS MA 3008 Y11817 recA
Neisseria polysaccharea NCTC 11858 057904 recA
Neisseria sicca NRL 30016 AJ223872 recA
Neisseria subflava NRL 30017 AJ223874 recA
Paracoccus denitrificansDSM 413 059631 recA
Pasteurella multocida X99324 recA
Porphyromonas gingivalisW83 070054 recA
Prevotella ruminicola JCM 8958 061227 recA
Proteus mirabilis pG1300 X14870 recA
Proteus vulgaris X55555 recA
Pseudomonas aeruginosa X05691 recA
Pseudomonas aeruginosa PAM 7 X52261 recA
Pseudomonas aeruginosa PA012 D13090 recA
Pseudomonas cepacia D90120 recA
Pseudomonas fluorescens OE 28.3 M96558 recA
Pseudomonas putida L12684 recA
Pseudomonas putida PpS145 070864 recA
Rhizobium leguminosarum VF39 X59956 recA
biovar viciae
Rhizobium phaseoli CNPAF512 X62479 recA
Rhodobacter capsulatus J50 X82183 recA
Rhodobacter sphaeroides 2.4.1 X72705 recA
Rhodopseudomonas palustrisN 7 D84467 recA
Rickettsia prowazekii Madrid E AJ235273 recA
Rickettsia prowazekii Madrid E 001959 recA
Serratia marcescens M22935 recA
Shigella flexneri X55553 recA
Shigella sonnei KNIH104S AF101227 recA
Sinorhizobium meliloti 2011 X59957 recA
Staphylococcus aureus L25893 recA
Streptococcus gordonii Challis V288 L20574 recA
Streptococcus mutans UA96 M81468 recA
Streptococcus pneumoniae 217307 recA
110
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Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
Streptococcus pneumoniae8800 234303 recA
Streptococcus pyogenes NZ131 U21934 recA
Streptococcus salivarius M94062 recA
subsp. thermophilus
Streptomyces ambofaciensDSM 40697 230324 recA
Streptomyces coelicolorA3(2) AL020958 recA
Streptomyces lividans TK24 X76076 recA
Streptomyces rimosus R6 X94233 recA
Streptomyces venezuelaeATCC10712 U04837 recA
Synechococcus sp. PR6 M29495 recA
Thermotoga maritima L23425 recA
Thermus aquaticus L20095 recA
Thermus thermophilus HB8 D17392 recA
Thiobacillus ferrooxidans M26933 recA
Treponema pallidum Nichols AE001243 recA
Vibrio anguillarum M80525 recA
Vibrio cholerae 017 X71969 recA
Vibrio cholerae 2740-80 U10162 recA
Vibrio cholerae 5698 L42384 recA
Vibrio cholerae M549 AF117881 recA
Vibrio cholerae M553 AF117882 recA
Vibrio cholerae M645 AF117883 recA
Vibrio cholerae M793 AF117878 recA
Vibrio cholerae M794 AF117880 recA
Vibrio cholerae M967 AF117879 recA
Xanthomonas citri XW47 AF006590 recA
Xanthomonas oryzae AF013600 recA
Xenorhabdus bovienii T228/1 U87924 recA
Xenorhabdus nematophilusAN6 AF127333 recA
Yersinia pestis 231 X75336 recA
Fungi, parasites, human and plants
Anabaena variabilis ATCC 29413 M29680 recA
Arabidopsis thaliana U43652 recA (Rad51)
Candida albicans U39808 recA (Dmc1)
Coprinus cinereus Okayama-7 U21905 recA (Rad51
)
Emericella nidulans 280341 recA (Rad51
)
Gallus gallus L09655 recA (Rad51)
Homo sapiens D13804 recA (Rad51)
Homo sapiens D63882 recA (Dmc1)
Leishmania major Friedlin AF062379 recA (Rad51)
Leishmania major Friedlin AF062380 recA (Dmc1
)
Mus musculus D58419 recA (Dmc1
)
Neurospora crassa 74-OR23-1A D29638 recA (Rad51)
Saccharomyces cerevisiae D10023 recA (Rad51)
Schizosaccharomyces pombe 222691 recA (Rad51
)
Schizosaccharomyces pombe972h- AL021817 recA (Dmc1)
Tetrahymena thermophila PB9R AF064516 recA (Rad51)
Trypanosoma brucei stock 427 Y13144 recA (Rad51
)
Ustilago maydis U62484 recA (Rad51
)
Xenopus laevis D38488 recA (Rad51)
Xenopus laevis D38489 recA (Rad51
)
111
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Table 11. Microbial species for which tuf and/or atpD and/or recA sequences
are available in
public databases (continued).
Species Strain Accession number Coding gene*
* tuf indicates tuf sequences
tuf (C) indicates tuf sequences divergent from main (usually A and B) copies
of the elongation factor-Tu
tuf (EF-1 ) indicates tuf sequences of the eukaryotic type (elongation factor
1 a)
tuf (M) indicates tuf sequences from organellar (mostly mitochondrial) origin
atpD indicates atpD sequences of the F-type
atpD (V) indicates atpD sequences of the V-Type
recA indicates recA sequences
recA (Rad51 ) indicates rad51 sequences or homologs
recA (Dmc1) indicates dmcl sequences or homologs
' Nucleotides sequences published in Arch. Microbiol. 1990 153:241-247
2 These sequences are from theTIGR database (http://www.tigr.org/tdb/tdb.html)
3 Nucleotides sequences published in FEMS Microbiology Letters 1988 50:101-106
112
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Table 12. Bacterial species used to test the specificity of the Staphylococcus
genus-specific
amplification primers derived from tuf sequences.
Strain Reference number Strain Reference number
Staphylococci species Other Gram-positive
(n=27) bacteria (n=20)
Staphylococcus arlettaeATCC 43957 Bacillus subtilis ATCC 27370
Staphylococcus aureusATCC 35844 Enterococcus avium ATCC 14025
subsp. anaerobius
ATCC 43300 Enterococcus durans ATCC 19432
Staphylococcus aureus
subsp. aureus
Staphylococcus auricularisATCC 33753 Enterococcus faecalis ATCC 19433
Staphylococcus capitisATCC 27840 Enterococcus faecium ATCC 19434
subsp. capitis
Staphylococcus capraeATCC 35538 Enterococcus flavescensATCC 49996
Staphylococcus carnosusATCC 51365 Enterococcus gallinarumATCC 49573
Staphylococcus chromogenesATCC 43764 Lactobacillus acidophilusATCC 4356
Staphylococcus cohniiDSM 20260 Lactococcus lactis ATCC 11454
subsp. urealyticum
Staphylococcus delphiniATCC 49171 Listeria innocua ATCC 33090
Staphylococcus epidermidisATCC 14990 Listeria ivanovii ATCC 19119
Staphylococcus equorumATCC 43958 Listeria monocytogenes ATCC 15313
Staphylococcus fells ATCC 49168 Macrococcus caseolyticusATCC 13548
Staphylococcus gallinarumATCC 35539 Streptococcus agalactiaeATCC 13813
Staphylococcus haemolyticusATCC 29970 Streptococcus anginosusATCC 33397
Staphylococcus hominisATCC 27844 Streptococcus bovis ATCC 33317
Staphylococcus hyicusATCC 11249 Streptococcus mutans ATCC 25175
Staphylococcus intermediusATCC 29663 Streptococcus pneumoniaeATCC 6303
Staphylococcus kloosisATCC 43959 Streptococcus pyogenes ATCC 19615
Staphylococcus lentusATCC 29070 Streptococcus salivariusATCC 7073
Staphylococcus lugdunensisATCC 43809
Staphylococcus saprophyticusATCC 15305
Staphylococcus schleiferiATCC 49545
subsp. coagulans
Staphylococcus sciuriATCC 29060
subsp. sciuri
Staphylococcus simulansATCC 27848
Staphylococcus warneriATCC 27836
Staphylococcus xylosusATCC 29971
Gram-negative bacteria
(n=33)
Acinetobacter baumanniiATCC 19606 Morganella morganii ATCC 25830
Bacteroides distasonisATCC 8503 Neisseria gonorrhoeae ATCC 35201
Bacteroides fragilis ATCC 25285 Neisseria meningitidis ATCC 13077
Bulkholderia cepacia ATCC 25416 Proteus mirabilis ATCC 25933
Bordetella pertussis ATCC 9797 Proteus vulgaris ATCC 13315
Citrobacter freundii ATCC 8090 Providencia rettgeri ATCC 9250
Enterobacter aerogenesATCC 13048 Providencia stuartii ATCC 29914
Enterobacter cloacae ATCC 13047 Pseudomonas aeruginosa ATCC 27853
Escherichia coli ATCC 25922 Pseudomonas fluorencensATCC 13525
Haemophilus influenzaeATCC 8907 Salmonella choleraesuisATCC 7001
Haemophilus parahaemolyticus Salmonella typhimurium ATCC 14028
ATCC 10014
Haemophilus parainfluenzaeATCC 7901 Serratia marcescens ATCC 8100
Hafnia alvei ATCC 13337 Shigella flexneri ATCC 12022
Kingella indologenes ATCC 25869 Shigella sonnei ATCC 29930
Klebsiella oxytoca ATCC 13182 Stenotrophomonas maltophiliaATCC 13843
Klebsiella pneumoniaeATCC 13883 Yersinia enterocoliticaATCC 9610
Moraxella catarrhalisATCC 25240
113
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Table 13. Bacterial species used to test the specificity of the penicillin-
resistant Streptococcus
pneumoniae amplification primers derived from pbpla sequences.
Strain Reference number Strain Reference number
Gram-positive species
(n=67)
Abiotrophia adiacens ATCC 49175 Staphylococcus hominisATCC 27844
Abiotrophia defectiva ATCC 49176 Staphylococcus lugdunensisATCC 43809
Actinomyces pyogenes ATCC 19411 Staphylococcus saprophyticusATCC 15305
Bacillus anthracis ATCC 4229 Staphylococcus simulansATCC 27848
Bacillus cereus ATCC 14579 Staphylococcus. warneriATCC 27836
Bifidobacterium breve ATCC 15700 Streptococcus acidominimusATCC 51726
Clostridium difficile ATCC 9689 Streptococcus agalactiaeATCC 12403
Enterococcus avium ATCC 14025 Streptococcus anginosusATCC 33397
Enterococcus casseliflavusATCC 25788 Streptococcus bovis ATCC 33317
Enterococcus dispar ATCC 51266 Streptococcus constellatusATCC 27823
Enterococcus durans ATCC 19432 Streptococcus cricetusATCC 19624
Enterococcus faecalis ATCC 29212 Streptococcus cristatusATCC 51100
Enterococcus faecium ATCC 19434 Streptococcus downei ATCC 33748
Enterococcus flavescensATCC 49996 Streptococcus dysgalactiaeATCC 43078
Enterococcus gallinarumATCC 49573 Streptococcus equi ATCC 9528
Enterococcus hirae ATCC 8043 Streptococcus ferns ATCC 33477
Enterococcus mundtii ATCC 43186 Streptococcus gordoniiATCC 10558
Enterococcus raffinosusATCC 49427 Streptococcus intermediusATCC 27335
Lactobacillus lactis ATCC 19435 Streptococcus mitis ATCC 903
Lactobacillus monocytogenesATCC 15313 Streptococcus mitis LSPQ 2583
Mobiluncus curtisii ATCC 35242 Streptococcus mitis ATCC 49456
Peptococcus niger ATCC 27731 Streptococcus mutans ATCC 27175
Pepfostreptococcus ATCC 6919 Streptococcus oralis ATCC 10557
scones
Peptostreptococcus ATCC 27337 Streptococcus oralis ATCC 9811
anaerobius
Peptostreptococcus ATCC 2639 Streptococcus oralis ATCC 35037
asaccharolyticus Streptococcus parasanguinisATCC 15912
Peptostreptococcus ATCC 51172 Streptococcus parauberisATCC 6631
lactolyticus
Peptostreptococcus ATCC 15794 Streptococcus rattus ATCC 15912
magnus
Peptostreptococcus ATCC 9321 Streptococcus salivariusATCC 7073
prevotii
Peptostreptococcus ATCC 35098 Streptococcus sanguinisATCC10556
tetradius
Staphylococcus aureus ATCC 25923 Streptococcus suis ATCC 43765
Staphylococcus capitisATCC 27840 Streptococcus uberis ATCC 19436
Staphylococcus epidermidisATCC 14990 Streptococcus vestibularisATCC 49124
Staphylococcus haemolyticusATCC 29970
Gram-negative species
(n=33)
Actinetobacter baumanniiATCC 19606 Moraxella morganii ATCC 13077
Bordetella pertussis ATCC 9797 Neisseria gonorrhoeae ATCC 35201
Citrobacter diversus ATCC 27028 Neisseria meningitidisATCC 13077
Citrobacter freundii ATCC 8090 Proteus mirabilis ATCC 25933
Enterobacter aerogenesATCC 13048 Proteus vulgaris ATCC 13315
Enterobacter agglomeransATCC 27155 Providencia alcalifaciensATCC 9886
Enterobacter cloacae ATCC 13047 Providencia rettgeri ATCC 9250
Escherichia coli ATCC 25922 Providencia rustigianiiATCC 33673
Haemophilus ducreyi ATCC 33940 Providencia stuartii ATCC 33672
Haemophilus haemolyficusATCC 33390 Pseudomonas aeruginosaATCC 35554
Haemophilus influenzaeATCC 9007 Pseudomonas fluorescensATCC 13525
Haemophilus parainfluenzaeATCC 7901 Pseudomonas stutzeri ATCC 17588
Hafnia alvei ATCC 13337 Salmonella typhimuriumATCC 14028
Klebsiella oxytoca ATCC 13182 Serratia marcescens ATCC 13880
Klebsiella pneumoniae ATCC 13883 Shigella flexneri ATCC 12022
Moraxella atlantae ATCC 29525 Yersina enterocoliticaATCC 9610
Moraxella catarrhalis ATCC 43628
114
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Table 14. Bacterial
species (n=104)
detected by the
platelet contaminants
assay. Bold characters
indicate the major acterial
b contaminants
found
in
platelet
concentrates.
Abiotrophia adiacens Klebsiella oxytoca Staphylococcus simulans
Abiotrophia defectiva Klebsiella pneumoniaeStaphylococcus warneri
Acinetobacter baumannii Legionella pneumophilaStenotrophomonas malfophilia
Acinetobacter Iwoffi Megamonas hypermegale80 Streptococcus acidominimus
Aerococcus viridans 45 Moraxella atlanfae Streptococcus agalactiae
10Bacillus anthracis Moraxella catarrhalisStreptococcus anginosus
Bacillus cereus Morganella morganii Streptococcus bovis
Bacillus subtilis Neisseria gonorrheae Streptococcus constellatus
Brucella abortus Neisseria meningitides85 Streptococcus cricetus
Burkholderia cepacia50 Pasteurella aerogenesStreptococcus cristatus
15Citrobacter diversus Pasfeurella multocidaStreptococcus dysgalactiae
Citrobacter freundii Pepfostreptococcus Streptococcus equi
magnus
Enterobacter aerogenes Proteus mirabilis Streptococcus ferns
Enferobacfer agglomerans Providencia alcalifaciens90 Streptococcus gordonii
Enterobacfer cloacae55 Providencia rettgeri Streptococcus intermedius
20Enferococcus avium Providencia rusfigianiiStreptococcus macacae
Enterococcus casseliflavus Providencia si'uartiiStreptococcus mites
Enterococcus dispar Pseudomonas aeruginosaStreptococcus mutans
Enterococcus durans Pseudomonas fluorescens95 Streptococcus oralis
Enterococcus faecalis60 Pseudomonas stutzeri Streptococcus parasanguinis
25Enterococcus faecium Salmonella bongori Streptococcus parauberis
Enterococcus flavescens Salmonella choleraesuisStreptococcus pneumoniae
Enterococcus gallinarum Salmonella enteritidisStreptococcus pyogenes
Enterococcus mundtii Salmonella gallinarum100 Streptococcus ratti
Enterococcus raffinosus65 Salmonella typhimuriumStreptococcus salivarius
30Enterococcus solitarius Serratia liquefaciensStreptococcus sanguinis
Escherichia coli Serratia marcescens Streptococcus sobrinus
Gemella morbillorum Shigella flexneri Streptococcus uberis
Haemophilus ducreyi Shigella sonnei 105 Streptococcus vesfibularis
Haemophilus haemolyticus70 Staphylococcus aureusVibrio cholerae
35Haemophilus influenzae Staphylococcus capifisYersinia enterocolitica
Haemophilus Staphylococcus epidermidisYersinia pestis
parahaemolyticus Staphylococcus haemolyticusYersinia pseudotuberculosis
Haemophilus parainfluenzae Staphylococcus hominis
Hafnia alvei 75 Staphylococcus lugdunensis
40Kingella kingae Staphylococcus saprophyficus
115
CA 02307010 2000-OS-19
Table 15. Microrganisms identified by automated systems.
Abiotrophia adiacens75 Alcaligenes xylosoxidans Brevibacterium species
(Streptococcus subsp. 1$~ Brevundimonas (Pseudomonas)
adjacens) xylosoxidans diminuta
Abiotrophia defectiva Alloiococcus otitis Brevundimonas (Pseudomonas)
(Streptococcus Anaerobiospirillum
succiniciproducens
defecfivus) Anaerovibrio lipolytica vesicularis
Achromobacterspecies
Acidaminococcus termentans8~ Arachnia propionica Brevundimonas species
155 Brochothrix thermosphacta
Acinetobacteralcaligenes Arcanobacterium (Actinomyces)
Acinetobacter anitratus bernardiae Brucella abortus
Acinetobacier baumannii Arcanobacterium (Actinomyces) Brucella canis
1~ Acinetobactercalcoaceticus pyogenes Brucella melitensis
Acinetobacter calcoaceticus85 Arcanobacterium haemolyticum Brucella ovis
biovar 16~ ecies
ll
B
anitratus Arcobacter cryaerophilus a sp
ruce
Acinetobacter calcoaceticus (Campylobacter cryaerophila) Brucella suis
biovar
Iwoffi Arthrobacterglobiformis Budvicia aquatica
Acinetobactergenomospecies Arthrobacterspecies Burkholderia
(Pseudomonas)
cepacia
Burkholderia (Pseudomonas)
gladioli
Acinetobacter haemolyticus9~ Anciozyma telluris 165 Burkholderia
(Pseudomonas)
(Torulopsis mallei
Acinetobacter johnsonii pintolopesii) Burkholderia (Pseudomonas)
ii Atopobium minutum
(Lactobacillus
Acinetobacter jun minutus) pseudomallei
2~ Acinetobacter Iwoffii Aureobacterium species Burkholderia species
Acinetobacter radioresistens
Acinetobacterspecies95 Bacillus amyloliquefaciens Buttiauxella agrestis
17~ r coli
t
l
b
Actinobacillus actinomycetemcomitans Bacillus anthracis ac
e
o
Campy
Actinobacillus capsulatus Bacillus badius Campylobacter concisus
Actinobacillus equuli Bacillus cereus Campylobacter fetus
Campylobacter fetus
subsp. fetus
Actinobacillus hominis1~~ Bacillus circulans Campylobacter fetus
lans subsp.
ill
Actinobacillus lignieresii us coagu 1~$ venerealis
Bac
Actinobacillus pleuropneumoniae Bacillus firmus Campylobacter
hyointestinalis
Actinobacillus species Bacillus lentus Campylobacter jejuni
Bacillus licheniformis subsp. doylei
Actinobacillus suis Bacillus megaterium Campylobacter jejuni
s ureae subsp. jejuni
ill
b
ti
A
u 1~5 Bacillus mycoides Campylobacter lari
ac
no
c
i
s Bacillus pantothenticus18~ Campylobacter lari
Actinomyces bov subsp. UPTC
Actinomyces israelii Bacillus pumilus Campylobacter mucosalis
i
Actinomyces meyer Bacillus species Campylobacterspecies
Actinomyces naeslundii
Actinomyces neuii Bacillus sphaericus Campylobactersputorum
subsp. anitratus lobacter sputorum
subsp.
Cam
Actinomyces neuii 11 Bacillus stearothermophilus185 py
subsp. neuii ~ l
b
b
Actinomyces odontolyticus Bacillus subtilis us
i u
i u
i Campylobacter sputorum
subsp.
Actinomyces pyogenes ens tecalis
s
ng
Bacillus thur
Actinomyces radingae Bacteroides caccae Campylobacter sputorum
ill subsp.
Actinomyces species osus
Bacteroides cap
Actinomyces turicensis115 Bacteroides distasonis sputorum
ii 19~ Campylobacter upsaliensis
Actinomyces viscosus Bacteroides eggerth Candida (Clavispora)
i Bacteroides fragilis lusitaniae
es Bacferoides merdae Candida (Pichia)
Aerococcus spec guilliermondii
Aerococcus viridans Bacteroides ovatus Candida (Torulopsis)
iae glabrata
Aeromonas cav 12~ Bacteroides species Candida albicans
Aeromonas hydrophila
Aeromonas hydrophila 8acteroides splanchnicus195 Candida boidinii
group
Aeromonas jandaei Bacferoides stercoris Candida catenulata
Aeromonas salmonicida Bacteroides thetaiotaomicron Candida ciferrii
Aeromonas salmonicida Bacteroides uniformis Candida colliculosa
subsp.
achromogenes 125 Bacteroides ureolyticus Candida conglobata
(B. corrodens)
Aeromonas salmonicida Bacteroides vulgatus2~~ Candida curvata
subsp. (Cryptococcus
masoucida Bergeyella (Weeksella) curvatus)
zoohelcum
Aeromonas salmonicida Bifidobacterium adolescentis Candida dattila
subsp.
5$ salmonicida Bifidobacterium bifidum Candida dubliniensis
Aeromonas schubertii13~ Bifidobacterium breve Candida famata
205 l
bosa
did
C
Aeromonas sobria Bifidobacterium dentium o
an
a g
Aeromonas species 8ifidobacterium infantis Candida hellenica
Aeromonas trota Bifidobacterium species Candida holmii
60 Aeromonas veronii Blasfoschizomyces Candida humicola
(Dipodascus)
Aeromonas veronii 135 capitatus Candida inconspicua
biovar sobria 21~ edia
i
t
did
Aeromonas veronii Bordetella avium erm
biovar veronii n
a
Can
Agrobacterium radiobacter Bordetella bronchiseptica Candida kelyr
Agrobacterium species Bordetella parapertussis Candida krusei
65 Agrobacterium fumefaciens Bordetella pertussis Candida lambica
Alcaligenes denitrificans14~ Candida magnoliae
Bordetella 215 marls
species did
C
Alcaligenes faecalis Borrelia species an
a
Alcaligenes odorans Branhamella (Moraxella) Candida melibiosica
catarrhalis
Alcaligenes odorans Branhamella species Candida membranaefaciens
(Alcaligenes
faecalis) Brevibacillus brevis Candida norvegensis
Alcaligenes species 145 Candida norvegica
Brevibacillus 22~ il
laterosporus sis
id
Alcaligenes xylosoxidans Brevibacterium caseio
a
paraps
Cand
AIcaIigenes xylosoxidans Brevibacterium epidermidis Candida
paratropicalis
subsp.
denitrificans Brevibacterium linens Candida pelliculosa
116
CA 02307010 2000-OS-19
Table 15. Microrganisms identified by automated systems (continued).
Candida pseudotropicalis Clostridium hastiforme Corynebacterium
urealyticum
(group
Candida pulcherrima 8~ Clostridium histolyticum D2)
Corynebacterium xerosis
Candida ravautii Clostridium innocuum16~ Crypfococcus albidus
Clostridium limosum
Candida rugasa Clostridium novyi Cryptococcus ater
Candida sake
Candida silvicola Clostridium novyi Cryptococcus cereanus
A
Candida species g$ Clostridium paraputrificum Cryptococcus gastricus
Candida sphaerica Clostridium perfringens Cryptococcus humicolus
165 lactativorus
t
Candida sfellatoidea Clostridium putrificum ococcus
Cryp
1~ Candida tenuis Clostridium ramosum Cryptococcus laurentii
Candida tropicalis Clostridium septicum Cryptococcus luteolus
Candida utilis 9~ Clostridium sordellii Cryptococcus melibiosum
Candida valida Clostridium species Cryptococcus neoformans
1~~ ecies
s s
t
C
Candida vini Clostridium sphenoides p
ococcu
ryp
1$ Candida viswanathii Clostridium sporogenes Cryptococcus terreus
l Cryptococcus uniguttulatus
i
Candida zeylanoides 95 e Debaryomyces hansenii
na
Clostridium subterm
rtium
t
idi
t
Cl
Capnocytophaga gingivalis um Debaryomyces marama
e
os
r
Clostridium tetani
Capnocytophaga ochracea Clostridium tyrobutyricum1~$ Debaryomyces
polymorphus
Capnocytophaga species i
2~ Capnocytophaga sputigena Comamonas (Pseudomonas) es
Debaryomyces spec
Dermabacter hominis
Cardiobacterium hominis1~~ acidovorans Dermacoccus (Micrococcus)
Comamonas (Pseudomonas)
Carnobacterium divergens testosteroni nishinomiyaensis
Carnobacferium piscicola
CDC group ED-2 Comamonas species 18~ Dietzia species
Edwardsiella hoshlnae
25 CDC group EF4 (Pasteurella Corynebacterium accolens Edwardsiella
ictaluri
sp.)
CDC group EF-4A 1~5 Corynebacterium afermentans Edwardsiella species
Corynebacterium amycolatum
CDC group EF-4B Corynebacterium aquaticum Edwardsiella farda
CDC group EQ-Z
CDC group HB-5 Corynebacterium argentoratenseIg5 Eikenella corrodens
i Empedobacter brevis
(Flavobacterium
3~ CDC group II K-2 s breve)
Corynebacterium aur
CDC group IV C-2 11~ Corynebacterium bovis Enterobacteraerogenes
(Bordetella-like) Corynebacteriumcoyleae
CDC rou M5
9 P Corynebacterium cystitidis Enterobacter agglomerans
CDC group M6
Cedecea davisae Corynebacterium diphtheriae190 Enterobacter amnigenus
35 Cedecea lapagei Corynebacterium diphtheriae Enterobacter
amnigenus
biotype asburiae
Cedecea neteri belfanti (CDC enteric group
115 e 17)
htheriae biot Enterobacter amnigenus
di biogroup 1
i
Cedecea species yp Enterobacter amnigenus
p biogroup 2
um
Corynebacter
Cellulomonas (Oerskovia) gravis 195 Enterobacter asburiae
turbata Corynebacterium diphtheriae
i biotype
es intermedius Enterobacter cancerogenus
Cellulomonas spec
Chlamydia species
Chromobacterium violaceum Corynebacterium diphtheriae Enterobacter
cloacae
biotype
Chryseobacterium I2~ mitis Enterobactergergoviae
(Flavobacterium)
indologenes Corynebacterium flavescens Enterobacter hormaechei
2~~ rmedius
i
t
t
b
Chryseobacterium Corynebacterium glucuronolyticum n
(Flavobacterium) er
e
ac
Entero
45 meningosepticum Corynebacterium glucuronolyticum- Enterobacter
sakazakii
Chryseobacterium seminale Enterobacter species
gleum 125 A Enterobacter taylorae
rou
i
t
b
Chryseobacterium p Enterobacter taylorae
species er (CDC enteric
l um g
ac
Coryne
Corynebacterium group
A-4
ogenes Corynebacterium group2~$ group 19)
Chryseomonas indo A-5
Citeromyces matritensis
$~ Citrobacteramalonaticus Corynebacterium group Enterococcus
(Streptococcus)
ANF
Citrobacter braakii Corynebacterium group cecorum
130 B Enterococcus (Streptococcus)
Corynebacterium group faecalis
B-3
Citrobacter diversus Corynebacterium group (Group D)
Citrobacter farmeri F
Citrobacter freundii Corynebacterium group21~ Enterococcus (Streptococcus)
F-1
$5 Citrobacter freundii Corynebacterium group faecium(Group D)
complex F-2 Enterococcus (Streptococcus)
Citrobacter koseri 135 Corynebacterium group saccharolyticus
kii G
Corynebacterium group
G-1
Citrobactersedla Corynebacterium group Enterococcus avium
Citrobacterspecies G-2 (Group D)
Citrobacter werkmanii Corynebacterium group215 Enterococcus casseliflavus
I
Citrobacter youngae Corynebacterium group (Steptococcus faecium
I-2 subsp.
Clostridium acetobutylicum Corynebacterium jeikeium casseliflavus)
(group JK) Enterococcus durans
(Streptococcus
Clostridium barati 14~ Corynebacterium kutscheri faecium subsp. durans)
(C. (Group D)
Clostridium beijerinckii murium) 220 Enterococcus gallinarum
t Corynebacterium macginleyi
f
65 ans Corynebacterium minutissimum Enterococcus hirae
ermen
Clostridium bi
Clostridium botulinum
Clostridium botulinum Corynebacterium pilosum Enterococcus malodoratus
(NP) B&F
Clostridium botulinum145 Corynebacterium propinquum Enterococcus mundtii
(NP) E
Clostridium botulinum Corynebacterium Enterococcus raffinosus
(P) A&H 225 ecies
occus s
t
E
Clostridium botulinum pseudodiphtheriticum p
(P) F eroc
n
Clostridium botulinum Corynebacterium pseudotuberculosis Envinia
amylovora
G1
Clostridium botulinumCorynebacterium Envinia carotovora
G2 pyogenes Erwinia carotovora
m subsp. atroseptica
renale
t
i
b
I5~
Clostridium butyricum er Envinia carotovora
i u subsp.
ac
Coryne
Corynebacterium renale
group
s Corynebacterium seminale230
Clostridium cadaver betavasculorum
Clostridium chauvoei
Clostridium clostridiiforme Corynebacterium species Envinia carotovora
subsp. carotovora
Clostridium difficile Corynebacterium striatum Envinia chrysanthemi
(C.
Clostridium fallax I5~ Envinia cypripedii
flavidum)
Clostridium glycolicum Corynebacterium ulcerans Erwinia mallotivora
11~
CA 02307010 2000-OS-19
Table 15. Microrganisms identified by automated systems (continued).
VII Lactobacillus paracasei
subsp.
Erwinia nigrifluens 8~ Haemophilus parainfluenzae paracasei
Erwinia quercina biotype
Envinia rhapontici VIII Lactobacillus pentosus
Haemophilus paraphrohaemolyticus16~ Lactobacillus plantarum
Erwinia rubrifaciens Haemophilus paraphrophilus Lactobacillus salivarius
li
i
i
i
c Haemophilus segnis Lactobacillus salivarius
s var. salicinlus
n
a sa
Erw
Erwinia species 85 Haemophilus somnus Lactobacillus species
Erysipelothrix rhusiopathiae i
Erysipelothrix species Haemophilus species s
165 Lactococcus diacitilact
arvieae
coccus
t
L
Escherichia blattae Hafnia alvei g
Hanseniaspora guilliermondii ac
o
Lactococcus lactis
subsp. cremoris
l~ Escherichia coli Hanseniaspora uvarum Lactococcus lactis
Escherichia coli A-D subsp. diacitilactis
Escherichia coli 0157:H79~ Hanseniaspora valbyensis Lactococcus lactis
subsp. hordniae
Escherichia fergusonii Hansenula anomala Lactococcus lactis
1~~ subsp. lactis
lantarum
Escherichia hermannii Hansenula holstii Lactococcus p
Lactococcus raffinolactis
1$ Escherichia species Hansenula polymorpha Leclercia adecarboxylata
Helicobacter (Campylobacter)
cinaedi
Escherichia vulneris 95 Helicobacter (Campylobacter) Legionella species
Eubacterium aerofaciens
Eubacterium alactolyticum fennelliae Leminorella species
i 1~5 Leptospira species
l
Eubacterium lenfum or Leptotrichia buccalis
Helicobacter (Campylobacter)
py
li
2~ Eubacterium limosum s Leuconostoc (Weissella)
Issatchenkia orienta
Eubacterium species 10~Kingella denitrificans paramesenteroides
enes
dolo
ll
i
Ki
Ewingella americana n Leuconostoc carnosum
g
a
nge
Kingella kingae
Filobasidiella neoformans Kingella species 1 Leuconostoc citreum
g~
25 Fllobasidium floriforme Klebsiella omithinolytica Leuconostoc gelidum
Fllobasidium uniguttulatum
Flavimonas oryzihabitans Klebsiella oxytoca Leuconosfoc lactis
Il)5ticola Leuconostoc mesenteroides
l
i
ll
b
Flavobacterium gleum an Leuconostoc mesenteroides
a p subsp.
s
e
Kle
Klebsiella pneumoniae
subsp.
Flavobacterium indologenes 185 cremoris
Flavobacterium odoratum ozaenae Leuconostoc mesenteroides
ecies Klebsiella pneumoniae subsp.
i subsp.
t
b
er dextranicum
um sp
Flavo
ac
Francisella novicida 11~pneumoniae Leuconostoc mesenteroides
Klebsiella pneumoniae subsp.
subsp.
Francisella philomiragia rhinoscleromatis mesenteroides
Francisella species Klebsiella species 19~ Leuconostoc species
Francisella tularensis
35 Fusobacterium mortiferum Klebsiella terrigena Listeria grayi
Fusobacterium necrogenes Kloeckera apiculata Listeria innocua
Fusobacterium necrophorum115Kloeckera apis Listeria ivanovii
Listeria monocytogenes
Fusobacterium nucleatum Kloeckera japonica 195 Listeria murrayi
Fusobacterium species Kloeckera species Listeria seeligeri
vera ascorbata
Klu
Fusobacterium varium y Lisferia species
i Kluyvera cryocrescens
es I2~Kluyvera species Listeria welshimeri
Gaffkya spec
Gardnerella vaginalis
Gemella haemolysans Kluyveromyces lactis Megasphaera elsdenii
i 2~~ Methylobacterium
mesophilicum
Gemella morbillorum anus Metschnikowia pulcherrima
Kluyveromyces manc
veromyces thermotolerans
Klu
45 Gemella species y Microbacterium species
Geotrichum candidum Kocuria (Micrococcus)
kristinae
Geotrichum fermentansI25Kocuria (Micrococcus) Micrococcus luteus
rosea
Geotrichum penicillarum Kocuria(Micrococcus) Micrococcus lylae
varians 21J5ecies
occus s
Mi
Geotrichum penicillatum Koserella trabulsii p
croc
Geotrichum species Kytococcus (Micrococcus) Mobiluncus curtisii
sedentarius
Gordona species Lactobacillus (Weissella) Mobiluncus mulieris
viridescens
Haemophilus aegyptius13~Lactobacillus A Mobiluncus species
Moellerella wisconsensis
Haemophilus aphrophilus Lactobacillus acidophilus21~ Moraxella (Branhamella)
catarrhalis
Haemophilus ducreyi Lactobacillus 8 Moraxella atlantae
Lactobacillus brevis
55 Haemophilus haemoglobinophilus Lactobacillus buchneri Moraxella bovis
Haemophilus haemolyticus135Lactobacillus casei Moraxella lacunata
Haemophilus influenzae Lactobacillus casei Moraxella nonliquefaciens
Haemophilus influenzae subsp. casei
biotype I
Haemophilus influenzae Lactobacillus casei 215 Moraxella osloensis
biotype II subsp. lactosus
Haemophilus influenzae Lactobacillus casei Moraxella phenylpyruvica
biotype III subsp. rhamnosus
Haemophilus influenzae Lactobacillus catenaformis Moraxella species
biotype IV anella morganii
Mor
Haemophilus influenzae14~Lactobacillus cellobiosus g
biotype V d Morganella morganii
subsp. morganii
Haemophilus influenzae es 220 Morganella morganii
biotype VI Lactobacillus collinoi subsp. sibonii
il
Haemophilus influenzae us Mycobacterium africanum
biotype VII Lactobacillus coproph
t
i
65 Haemophilus influenzae us Mycobacterium asiaticum
biotype VIII spa
Lactobacillus cr
t
Haemophilus paragallinarum145us Mycobacterium avium
Lactobacillus curva
Lactobacillus delbrueckii
subsp.
Haemophilus parahaemolyticus bulgaricus Mycobacterium bovis
fl
uenzae Lactobacillus delbrueckii225
Haemophilus parain subsp. Mycobacterium
Haemophilus parainfluenzae chelonae
biotype I
Haemophilus delbrueckii Mycobacterium
fortuitum
parainfluenzae
biotype
II
Haemophilus parainfluenzae Lactobacillus delbrueckii Mycobacterium
gordonae
biotype subsp. lactis
I 150 Mycobacterium kansasii
Lactobacillus
fermentum
II Lactobacillus fructivorans Mycobacterium malmoense
Haemophilus parainfluenzae 230 terium marinum
biotype b
M
IV Lactobacillus helveticus ac
yco
7$ Lactobacillus helveticus Mycobacterium phlei
Haemophilus subsp. jugurti
parainfluenzae
biotype
V
Haemophilus parainfluenzae Lactobacillus jensenii Mycobacterium
scrofulaceum
biotype ti
VI 155 s
Lactobacillus Mycobacterium smegma
lindneri
Haemophilus parainfluenzae Lactobacillus minutus Mycobacterium species
biotype
118
CA 02307010 2000-OS-19
Table 15. Microrganisms identified by automated systems (continued).
Mycobacterium tuberculosis Pichia fermentans Saccharomyces exiguus
Saccharomyces kluyverii
Mycobacterium ulcerans80 Pichia membranaefaciens Saccharomyces species
i
Mycobacterium xenopi s 160 Sakaguchia dacryoides
Pichia norvegens
Pichia ohmeri
$ Mycoplasma fermentans Pichia spartinae (Rhodosporidium
dacryoidum)
Mycoplasma hominis
Mycoplasma orate Pichia species Salmonella arizonae
i
Mycoplasma pneumoniaeg5 Plesiomonas shigelloides s
Salmonella choleraesu
Salmonella enteritidis
Mycoplasma species Porphyromonas asaccharolytica165 Salmonella gallinarum
i Porphyromonas endodontalis
es Porphyromonas gingivalis Salmonella paratyphi
Myroides spec A
cinerea
i
N
i
sser Porphyromonas levee Salmonella paratyphi
a B
e
Neisseria elongata
subsp. elongata
Neisseria flava 90 Prevotella (Bacteroides) Salmonella pullorum
buccae
Neisseria flavescens Prevotella (Bacteroides) Salmonella species
buccalis 1~0 hi
ll
t
Neisseria gonorrhoeae Prevotella (Bacteroides) yp
corporis a
Salmone
Neisseria lactamica Prevotella (Bacteroides) Salmonella typhimurium
denticola
Neisseria meningitides Prevotella (Bacteroides) Salmonella typhisuis
loescheii
Neisseria mucosa 95 Prevotella (Bacteroides) SalmonellalArizona
orates
Neisseria perflava Prevotella (Bacteroides)disiens Serratia ficaria
1~'JSerratia fonticola
Neisseria polysaccharea Prevotella (Bacteroides)oris Serratia grimesii
Prevotella bivia
(Bacteroides bivius)
Neisseria saprophytes Prevotella intermedia Serratia liquefaciens
(Bacteroides
Neisseria sicca 100 intermedius) Serratia marcescens
Neisseria subflava
Neisseria weaveri Prevotella melaninogenica Serratia odorifera
180 Serratia odorifera
type 1
Neisseria weaveri (Bacteroides melaninogenicus) Serratia odorifera
(CDC group M5) type 2
Nocardia species PrevoteUa ruminicola Serratia plymuthica
Ochrobactrum anthropi105 Propionibacterium Serratia proteamaculans
i acnes
Propionibacterium
avidum
es Propionibacterium Serratia proteamaculans
Oerskovia spec granulosum subsp.
Oerskovia xanthineolytica
Oligella (Moraxella) Propionibacterium 185 proteamaculans
urethralis propionicum Serratia proteamaculans
i subsp.
Oligella species es
Propionibacterium
spec
Oligella ureolytica Proteus mirabilis quinovora
110 enneri Serratia rubidaea
P
f
Paenibacillus alvei eus p Serratia species
ro
i
Paenibacillus macerans es 190 Shewanella (Pseudomonas,
Proteus spec
Proteus vulgaris
Paenibacillus polymyxa Prototheca species Alteromonas) putrefaciens
lomerans
t
P
oea agg Prototheca wickerhamii Shigella boydii
an
Pantoea ananas (Envinia
uredovora)
Pantoea dispersa 115 Prototheca zopfii Shigella dysenteriae
Pantoea species Providencia alcalifaciens Shigella flexneri
195 nnei
ll
Shi
Pantoea stewartii Providencia heimbachae ge
a so
Pasteurella (Haemophilus) Providencia rettgeri Shigella species
avium ii Sphingobacterium
multivorum
Pasteurella aerogenes120 Providencia rustigian Sphingobacterium
Providencia species species
Pasteurella gallinarum Providencia stuartii Sphingobacterium
Pasteurella haemolytica spiritivorum
Pasteurella haemolyticus Providencia stuartii200 Sphingobacterium
urea + thalpophilum
hingomonas (Pseudomonas)
S
Pasteurella multocida Pseudomonas (Chryseomonas) p
paucimobilis
Pasteurella multocida12$ luteola Sporidiobolus salmonicolor
SF Pseudomonas acidovorans
Pasteurella multocida Pseudomonas aeruginosa Sporobolomyces roseus
subsp.
multocida Pseudomonas alcaligenes205 Sporobolomyces salmonicolor
Pasteurella multocida
subsp. septica
Pasteurella pneumotropica Pseudomonas cepacia Sporobolomyces species
P lococcus (Peptococcus)
i Staph
Pasteurella species 130 . y
s ( saccharolyticus
Pseudomonas chlororaph
i
f
Pasteurella ureae ens) Staphylococcus arlettae
Pediococcus acidilactici ac
aureo
Pseudomonas fluorescens
Pediococcus damnosus Pseudomonas fluorescens210 Staphylococcus aureus
group Staphylococcus aureus
i (Coagulase-
Pediococcus pentosaceus na negative)
Pseudomonas mendoc
li
Pediococcus species 135 genes Staphylococcus auricularis
Pseudomonas pseudoalca
Pseudomonas putida
Peptococcus niger Pseudomonas species Staphylococcus capitis
i
es Pseudomonas stutzeri215 Staphylococcus capitis
Peptococcus spec subsp. capitis
Peptostreptococcus
anaerobius
Peptostreptococcus Pseudomonas testosterone Staphylococcus capitis
asaccharolyticus subsp.
Peptostreptococcus Pseudomonas vesicularis ureolyticus
indolicus
Peptostreptococcus 140 Pseudoramibacter Staphylococcus caprae
magnus (Eubacterium)
Peptostreptococcus alactolyticus Staphylococcus camosus
micros ll 220 Staphylococcus caseolyticus
Peptostreptococcus a) Staphylococcus chromogenes
parvulus Psychrobacter (Moraxe
Peptostreptococcus phenylpyruvicus Staphylococcus cohnii
prevotii
Peptostreptococcus Rahnella Staphylococcus cohnii
productus aquatilis subsp. cohnii
i 145
s Ralstonia
(Pseudomonas,
e Burkholderia) pickettii Staphylococcus cohnii
Peptostreptococcus subsp.
spec
Peptostreptococcus
tetradius
Phaecoccomyces exophialiae Rhodococcus (Corynebacterium)2~5
equi urealyticum
i Staphylococcus
epidermidis
Photobacterium es Staphylococcus
equorum
damselae Rhodococcus spec
l Rhodosporidium toruloides
a 150 Staphylococcus gallinarum
Pichia (Hansenula) Rhodotorula
anoma glutinis
ii
Pichia (Hansenula) Rhodotorula minuta Staphylococcus haemolyticus
jadin
Pichia (Hansenula) Rhodotorula mucilaginosa230
petersonii (R. rubra) Staphylococcus
Pichia angusta (Hansenula hominis
polymorpha) Rhodotorula species Staphylococcus
hominis
subsp.
Pichia carsonii (P. Rickettsia hominis
vim) species Staphylococcus hominis
ntocariosa subsp.
d
thi
155
R
Pichia etchellsii e novobiosepticus
a
o
i
i
Pichia farinosa ae
s
Saccharomyces cerev
119
CA 02307010 2000-OS-19
Table 15. Microrganisms identified by automated systems (continued).
60 Streptococcus Gamma Tetragenococcus
(non)- (Pediococcus)
Staphylococcus hyicus hemolytic 120 halophilus
Staphylococcus infermedius Streptococcus gordonii Torulaspora
delbrueckii
Staphylococcus kloosii Streptococcus Group (Saccharomyces
$ S B roses)
taphylococcus lentus Streptococcus Group Torulopsis candida
C
Staphylococcus lugdunensis6$ Streptococcus Group Torulopsis haemulonii
D
Staphylococcus saprophyticus Streptococcus Group12$ Torulopsis inconspicua
E
Staphylococcus schleiferi Streptococcus Group Treponema species
F
Staphylococcus sciuri Streptococcus Group Trichosporon asahii
G
Staphylococcus simulans Streptococcus Group Trichosporon asteroides
L
Staphylococcus species70 Streptococcus Group Trichosporon beigelii
P
Staphylococcus warneri Streptococcus Group130 Trichosporon cutaneum
U
Staphylococcus xylosus Streptococcus intermedius Trichosporon inkin
Stenotrophomonas Streptococcus intermedius Trichosporon mucoides
1$ (Xanthomonas)
l
ma (Streptococcus millers Trichosporon ovoides
tophilia II)
Stephanoascus ciferrii~$ Streptococcus intermedius Trichosporon pullulans
(viridans
Stomatococcus mucilaginosus Streptococcus) 13$ Trichosporon species
Streptococcus acidominimus Streptococcus millers Turicella otitidis
group
Streptococcus agalactiae Streptococcus mitis Ureaplasma species
S
treptococcus agalactiae Streptococcus mitis Ureaplasma urealyticum
(Group B) (viridans
Streptococcus agalactiae80 Streptococcus) Veillonella parvula
hemolytic (V. alcalescens)
Streptococcus agalactiae Streptococcus mitis140 Veillonella species
non- group
hemolytic Streptococcus mutans Vibrio alginolyticus
Streptococcus alactolyticus Streptococcus mutans Vibrio cholerae
2$ S (viridans
treptococcus anginosus Streptococcus) Vibrio damsels
Streptococcus anginosusg$ Streptococcus oralis Vibrio fluvialis
(Group D,
nonenterococci) Streptococcus parasanguis14$ Vibrio furnissii
Streptococcus beta-hemolytic Streptococcus pneumoniae Vibrio harveyi
group A
Streptococcus beta-hemolytic Streptococcus porcinus Vibrio hollisae
non-
A
B
group Streptococcus pyogenes Vibrio metschnikovii
or
Streptococcus beta-hemolytic90 Streptococcus pyogenes Vibrio mimicus
non- (Group A)
group A Streptococcus salivarius1$0 Vibrio parahaemolyticus
Streptococcus beta-hemolytic Streptococcus salivarius Vibrio species
(viridans
Streptococcus bovis Streptococcus) Vibrio species
3$ (Group D, SF
t
nonen Streptococcus salivarius Vibrio vulnificus
erococci) subsp.
Streptococcus bovis 9$ salivarius Weeksella (Bergeylla)
I virosa
Streptococcus bovis Streptococcus salivarius1$$ Weeksella species
II subsp.
Streptococcus canis thermophilus Weeksella virosa
Streptococcus constellatus Streptococcus sanguis Williopsis (Hansenula)
S saturnus
treptococcus constellatus Streptococcus sanguis Xanthomonas campestris
I (viridans
(Streptococcus millers100 Streptococcus) Xanthomonas species
I)
Streptococcus constellatus Streptococcus sanguis160 Yarrowia (Candida)
(viridans II lipolytica
Streptococcus) Streptococcus sanguis Yersinia aldovae
II (viridans
Streptococcus downei Streptococcus) Yersinia enterocolitica
4$ S
treptococcus dysgalactiae Streptococcus sobrinus Yersinia
enterocolitica
subsp. group
dysgalactiae 10$ Streptococcus species Yersinia frederiksenii
Streptococcus dysgalactiae Streptococcus suss 16$ Yersinia intermedia
subsp. I
equisimilis Streptococcus suss Yersinia intermedius
II
Streptococcus equi Streptococcus uberis Yersinia kristensenii
$0 (Group C/Group G
S
treptococcus) Streptococcus uberis Yersinia pestis
(viridans
Streptococcus equi 1 Streptococcus) Yersinia pseudotuberculosis
subsp. equi 10
Streptococcus equi Streptococcus vestibularis1 Yersinia
pseudotuberculosis
subsp. ~0 SF
zooepidemicus Streptococcus zooepidemicus Yersinia ruckeri
Streptococcus equinus Streptococcus zooepidemicus Yersinia species
$$ (Group
Streptococcus equinus C) Yokenella regensburgei
(Group D,
nonenterococci) 11$ Streptomyces somaliensis Yokenella regensburgei
(Koserella
Streptococcus equisimilis Streptomyces species1 trabulsiQ
~
$
Streptococcus equisimulis Suttonella (Kingella) Zygoascus hellenicus
(Group indologenes
C/Group G Streptococcus) Tatumella ptyseos Zygosaccharomyces
species
120
<IMG>
<IMG>
CA 02307010 2000-OS-19
C
O
L
1
N
N r
L
L Q
M
V N N 00 f~ d'
Q p
r NI N
N N ~
r
O U
a
=
N _
r
O
U
Q
M Q N
N L1J O
T 0~0
~ N
L
Q Q ~ N p
V ~ ~L ~L
o a a.
c~
T a~
O
T
L N N
N r
a1
123
CA 02307010 2000-OS-19
Annex I: Specific and ubiquitous primers for nucleic acid
amplification (tuf seguences).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
NO. position
10Bacterial pecies: Chlamyd3a pneumoniae
s
630 5' -CGGAGCTAT CCT CGTTTCA 20 2- 23
AGT
629a 5' -AAGTTCCAT CTC AAGGTCAAT A 20 146 -170
AAC
15Bacterial ecies: Chlamydia trachomatis
sp
554 5' -GTTCCTTAC ATC GTTTTTCTC 22 82- 105
GTT
555a 5' -TCTCGAACT TTC ATGTATGCA 22 249 -272
TCT
20Parasitical species : Cryptosporidium parvum
798 5' -TGGTTGTCC CAG ATCGTTT 865 158 -179
CCG
804a 5' -CCTGGGACG GCC GGCAT 865 664 -683
TCT
25799 5' -ACCTGTGAA TAC CAATCT 865 280 -300
AAG
805a 5' -CTCTTGTCC ATC GCAGT 865 895 -914
TTA
800 5' -GATGAAATC TTC GAAGTTGAT 865 307 -330
AAC
806a 5' -AGCATCACC AGA GATAAG 865 946 -966
CTT
30
801 5' -ACAACACCG AGA TCCCA 865 353 -372
AGA
803a 5' -ACTTCAGTG GTA CCAGC 865 616 -635
ACA
802 5' -TTGCCATTT CTG TCG'rT 865 377 -396
GTT
35807a 5' -AAAGTGGCT TCA GTTGC 865 981- 1000
AAG
Bacterial species: Ne3sseria gonorrhoeae
551 5'-GAA GAA 126 256 -280
AAA
ATC
TTC
GAA
CTG
GCT
A
40552a 5'-TAC ACG GCC GGT GAC TAC G 126 378 -396
Bacterial pecies: Streptococcus agalactiae
s
549 5'-GAA CGT GAT ACT GAC AAA CCT TTA 207-210b 308- 331c
45550a 5'-GAA GAA GAA CAC CAA CGT TG 207-210b 520- 539c
Bacterial ecies: Streptococcus pyogenes
sp
999 5'-TTG ACC TTG TTG ATG ACG AAG AG 1002 143 -165
$01000a 5'-TTA GTG TGT GGG TTG ATT GAA CT 1002 622 -644
1001 5'-AAG AGT TGC TTG AAT TAG TTG AG 1002 161 -183
1000a 5'-TTA GTG TGT GGG TTG ATT GAA CT 1002 622 -644
a These sequences fromthe complementary DNA of the of
are strand sequence the
originating fragmentgiven in the Sequence Listing.
b These sequences aligned to derive the corresponding
were primer.
c The nucleotide positions refer to the S. agalactiaetuf sequence
agment
fr
60(SEQ ID NO. 209).
124
CA 02307010 2000-OS-19
Annex I: Specific and for nucleic acid
ubiquitous primers
amplification (tuf sequences)
(continued).
OriginatingDNA
fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
ParasitiCal species: Trypanosome bruCel
820 5'-GAA GGA GGT GTC TGC TTA CAC 864 513 -533
821a 5'-GGC GCA AAC GTC ACC ACA TCA 864 789 -809
820 5'-GAA GGA GGT GTC TGC TTA CAC 864 513 -533
822a 5'-CGG CGG ATG TCC TTA ACA GAA 864 909 -929
Parasitical species: Trypanosome cruzi
794 5'-GAC GAC AAG TCG GTG AAC ~rT 840-842b 281- 300
795a 5'-ACT TGC ACG CGA TGT GGC AG 840-842b 874- 893c
Bacterial aenus: Bordetella sp.
825 5'-ATG AGC ARC GSA ACC ATC GTT CAG 863 1- 26
TG
826 5'-TCG ATC GTG CCG ACC ATG TAG AAC 863 1342 -1367
GC
Fungal genus: Candida sp.
576 5'-AAC TTC RTC AAG AAG GTY GGT TAC 407-426, 332- 3574
AA
428-432b
632a 5'-CCC TTT GGT GGR TCS TKC TTG GA 407-426, 791- 8134
428-432b
631 5'-CAG ACC AAC YGA IAA RCC ATT RAG 407-426, 523- 5484
AT
428-432b
632a 5'-CCC TTT GGT GGR TCS TKC TTG GA 407-426, 791- 8134
428-432b
633 5'-CAG ACC AAC YGA IAA RCC ITT RAG 407-426, 523- 5484
AT
428-432b
632a 5'-CCC TTT GGT GGR TCS TKC TTG GA 407-426, 791- 8134
428-432b
a These sequences are the complementary DNA of
from strand of the sequence the
originating fragment
given in the Sequence
Listing.
b These sequences were
aligned to derive the
corresponding primer.
c The nucleotide positionsrefer to the T. cruzi sequence SEQ
tuf fragment ID
(
N0. 842).
d The nucleotide positionsrefer to the C. albicans agment
tuf(EF-1) sequence fr
(SEQ ID N0. 408).
125
CA 02307010 2000-OS-19
Annex I: Specific and ubiguitous primers for nucleic acid
amplification (tuf sequences) (continued).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
NO. position
Bacterial enus: Clostridium sp.
g
796 5'-GGT CCA ATGCCW CAA AGA 32,719- 32 -52b
ACW
724,736a
797 5'-CAT TAA GAATGG YTT TGT SKC TCT 32,719- 320-346b
ATC
724,736a
808 5'-GCI TTA IWRGCA TTA RAY CCA 32,719- 224-247b
GAA
724,736a
809c 5'-TCT TCC TGTWGC AAC TCC TCT 32,719- 337-360b
TGT
724,736a
810 5'-AGA GMW ACAGAT AAR TTC TTA 32,719- 320-343b
SCA
724,736a
811c 5'-TRA ART AGAATT GTG TRT ATC C 32,719- 686-710b
GTC
724,736a
Bacterial enus: Corynebacterium
g sp.
545 5'-TAC ATC CTBGTY GCI AAC AAG TG 34-44,662a89- 1144
CTI
546c 5'-CCR CGI CCGGTR ATG AAG AT 34-44,662a350-3724
GTG
Parasitical genus: Entamoeba .
sp
703 5'-TAT GGA AATTCG AAA CT 512 38 -57
CAT
704c 5'-AGT GCT CCAATT AAT GG 512 442 -461
GTT
703 5'-TAT GGA AATTCG AAA CT 512 38 -57
CAT
705c 5'-GTA CAG TTCCAA TAC AA 512 534 -553
CTG
703 5'-TAT GGA AATTCG AAA CT 512 38 -57
CAT
706c 5'-TGA AAT CTTCAC ATC CA 512 768 -787
CAA
793 5'-TTA TTG TTGCTG CTG CT 512 149 -168
GTA
704c 5'-AGT GCT CCAATT AAT GG 512 442 -461
GTT
a These sequences alignedto derive e corresponding primer.
were th
b The nucleotide ionsrefer to the C. perfringenstuf sequence
posit fragment
(SEQ ID NO. 32).
c These sequences fromthecomplementary of the of
are DNA strand sequence the
S0 originating fragmentgiven the Sequence
in Listing.
d The nucleotide ionsrefer to the C. diphtheriaetuf sequence
posit fragment
(SEQ ID N0. 662).
126
CA 02307010 2000-OS-19
Annex I: Specific and ubiguitous primers for nucleic acid
amplification (tuf sequences) (continued).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
NO. position
Bacterial enus: Eaterococcus sp.
g
656 5'-AAT TAATGG CTGCAG AYG A 58-72a 273- 294b
TTG
657c 5'-TTG TCCACG TTCGAT TTC A 58-72a 556- 577b
RTC
656 5'-AAT TAATGG CTGCAG AYG A 58-72a 273- 294b
TTG
271c 5'-TTG TCCACG TTGGAT TTC A 58-72a 556- 577b
RTC
1137 5'-AAT TAATGG CTGCWG AYG AA 58-72a 273- 295b
TTG
1136c 5'-ACT TGTCCA CGTTSG TCT 58-72a 559- 579b
ATR
Parasitic al genus: Gisrdi s sp.
816 5'-GCT ACGACG AGATCA GC 513 305 -324
AGG
819c 5'-TCG AGCTTC TGGAGG AG 513 895 -914
AAG
817 5'-TGG AAGAAG GCCGAG TT 513 355 -374
GAG
818c 5'-AGC CGGGCT GGATCT TC 513 825 -844
TCT
Parasitical genus: Leishmania
sp.
701 5'-GTG TTCACG ATCATC GCG 514-526a 94- 1144
GAT
702c 5'-CTC TCGATA TCCGCG CG 514-526a 913- 932d
AAG
Bacterial enus: Staphylococcus
g sp.
553 5'-GGC CGTGTT GAACGT CAA ATC 176-203a 313- 337e
GGT A
575c 5'-TIA CCATTT CAGTAC CTG GTA 176-203a 653- 677e
CTT A
553 5'-GGC CGTGTT GAACGT CAA ATC 176-203a 313- 337e
GGT A
707c 5'-TWA CCATTT CAGTAC CTG GTA 176-203a 653- 677e
CTT A
Bacterial enus: Streptococcus
g sp.
547 5'-GTA CAGTTG CTTCAG GTA TC 206-231a 372- 394f
GAC
548c 5'-ACG TTCGAT TTCATC TTG 206-231a 548- 568f
ACG
a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the E. durans tuf sequence fragment (SEQ
ID
N0. 61).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
d The nucleotide positions refer to the L. tropica tuf(EF-1) sequence fragment
(SEQ ID N0. 526).
a The nucleotide positions refer to the S. aureus tuf sequence fragment (SEQ
ID
NO. 179).
f The nucleotide positions refer to the S. agalactiae tuf sequence fragment
(SEQ ID NO. 209).
127
CA 02307010 2000-OS-19
Annex I: Specific and for nucleic acid
ubiguitous
primers
amplification
(tuf seguences)
(continued).
Originating
DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
NO. position
10Parasitical Trypanosome sp.
genus:
823 5'-GAG CGGTAT GAY GAG ATT GT 529,840- 493- 512b
842,864a
824c 5'-GGC TTCTGC GGC ACC ATG CG 529,840- 1171-1190b
842,864a
Bacterial family: Mycobacteriaceae
539 5'-CCI TACATC CTB GTY GCI CTI AAC 122 85- 111
AAG
20540c 5'-GGD GCITCY TCR TCG WAI 'PCC TG 122 181 -203
Bacterial croup: Enterobacteriaceae group
933 5'-CAT CATCGT ITT CMT GAA CAA RTG 78,103,146,390- 4134
2$ 168,238,698a
934c 5'-TCA CGYTTR RTA CCA CGC AGI AGA 78,103,146,831- 8544
168,238,698a
Parasitical Trypanosomatidae family
family:
30
923 5'-GAC GCIGCC ATC CTG ATG ATC 511,514-526,166- 188e
529,840-842,
864a
924c 5'-ACC TCAGTC GTC ACG TTG GCG 511,514-526,648- 668e
35 529,840-842,
864a
925 5'-AAG CAGATG GTT GTG TGC TG 511,514-526,274- 293e
529,840-842,
40 864a
926c 5'-CAG CTGCTC GTG GTG CAT CTC GAT 511,514-526,676- 699e
529,840-842,
864a
a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the T. brucei tuf (EF-1) sequence fragment
(SEQ ID NO. 864).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
d The nucleotide positions refer to the E. coli tuf sequence fragment (SEQ ID
NO. 698).
a The nucleotide positions refer to the L. tropica tuf sequence fragment (SEQ
ID N0. 526).
128
CA 02307010 2000-OS-19
Annex I: Specific and ubiquitous primers for nucleic acid
amplification (tuf sequences) (continued).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Parasitical familv: Trypanosomatidae family (continued)
927 5'-ACG CGG AGA AGG TGC GCT T 511,514-526, 389-407b
529,840-842,
864a
928c 5'-GGT CGT TCT TCG AGT CAC CGC A 511,514-526, 778-799b
529,840-842,
864a
Bacterial group: Pseudomonads group
541 5'-GTK GAAATG TTC AAGCTGCT 153-155a 476-498d
CGC
542c 5'-CGG AARTAG AAC GGACGGTAG 153-155a 679-7024
TGS
541 5'-GTK GAAATG TTC AAGCTGCT 153-155a 476-4984
CGC
25544c 5'-AYG TTGTCG CCM ATTMCCAT 153-155a 749-7714
GGC
Universal primers
636 5'-ACT GGYGTT GAI TTCCGYAA 7,54,78, 470 -492e
ATG
100,103,159,
209,224,227b
637a 5'-ACG TCAGTI GTA AARTAGAA 7,54,78, 692 -714e
CGG
100,103,159,
209,224,227b
638 5'-CCA ATGCCA CAA CGTGARCAC 7,54,78, 35 -60f
ACI AT
100,103,159,
209,224,227b
639a 5'-TTT ACGGAA CAT WACACCWGT A 7,54,78, 469 -496f
TTC IAC
100,103,159,
209,224,227b
a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the L. tropica tuf (EF-1) sequence
fragment
(SEQ ID N0. 526).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
d The nucleotide positions refer to the P. aeruginosa tuf sequence fragment
(SEQ ID N0. 153).
a The nucleotide positions refer to the E. coli tuf sequence fragment (SEQ ID
N0. 78).
f The nucleotide positions refer to the B. cereus tuf sequence fragment (SEQ
ID
NO. 7).
129
CA 02307010 2000-OS-19
Annex I: Specific and ubiquitous primers for nucleic acid
amplification (tuf sequences) (continued).
Originating DNA fragment
$ SEQ ID NO. Nucleotide sequence SEQ ID Nucleotide
N0. position
643 5'-ACT GGI GTI GAR ATG TTC CGY AA 1,3,4,7,12, 470-492b
13,16,49,54,
72,78,85,88,
91,94,98,103,
108,112,115,
1$ 116,120,121,
126,128,134,
136,146,154,
159,179,186,
205,209,212,
224,238a
644c 5'-ACG TCI GTI GTI CKG AAR TAG AA 1,3,4,7,12, 692-714b
13,16,49,54,
72,78,85,88,
91,94,98,103,
2$ 108,112,115,
116,120,121,
126,128,134,
136,146,154,
159,179,186,
205,209,212,
224,238a
a These sequences were aligned to derive the corresponding primer.
3$ b The nucleotide positions refer to the E. coli tuf sequence fragment (SEQ
ID
N0. 78).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
130
CA 02307010 2000-OS-19
Annex I: Specific and ubiquitous primers for nucleic acid
amplification (tuf sequences) (continued).
Originating
DNA fragment
SEQ ID NO. Nucleotide sequence SEQ ID Nucleotide
N0. position
1~ 643 5'-ACT GGI GTIGAR ATGTTCCGYAA 1,3,4,7,12, 470-492b
13,16,49,54,
72,78,85,88,
91,94,98,103,
108,112,115,
1$ 116,120,121,
126,128,134,
136,146,154,
159,179,186,
205,209,212,
20 224,238a
645c 5'-ACG TCI GTIGTI CKGAARTARAA 1,3,4,7,12, 692-714b
13,16,49,54,
72,78,85,88,
91,94,98,103,
ZS 108,112,115,
116,120,121,
126,128,134,
136,146,154,
159,179,186,
3~ 205,209,212,
224,238a
646 5'-ATC GAC AAGCCI TTCYTIATGSC 2,13,82 317-339d
122,145a
35 647c 5'-ACG TCC GTSGTR CGGAAGTAGAAC 2,13,82 686-7114
TG
122,145a
646 5'-ATC GAC AAGCCI TTCYTIATGSC 2,13,82 317-339d
122,145a
40 648c 5'-ACG TCS GTSGTR CGGAAGTAGAAC 2,13,82 686-711d
TG
122,145a
45 a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the E. coli tuf sequence fragment (SEQ ID
N0. 78).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
50 d The nucleotide positions refer to the A. meyeri tuf sequence fragment
(SEQ ID
N0. 2)
131
CA 02307010 2000-OS-19
Annex I: Specific and ubiquitous primers for nucleic acid
amplification (tuf seguences) (continued).
Originating DNA fragment
S SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Universal primers
(continued)
649 5'-GTC CTA TGCCTC ARACWCGIGAGC 8,86,141,143a33- 58b
AC
650c 5'-TTA CGG AACATY TCAACACCIGT 8,86,141,143a473-495b
1S 636 5'-ACT GGY GTTGAI ATGTTCCGYAA 8,86,141,143a473-495b
651c 5'-TGA CGA CCACCI TCYTCYTTYTTC 8,86,141,143a639-663b
A
Sequencing
primers
556 5'-CGG CGC NATCYT SGTTGTTGC 6684 306 -326
557c 5'-CCM AGG CATRAC CATCTCGGTG 668d 1047-1068
694 5'-CGG CGC IATCYT SGTTGTTGC 6684 306 -326
557c 5'-CCM AGG CATRAC CATCTCGGTG 668d 1047-1068
2S
664 5'-AAY ATG ATIACI GGIGCIGCICAR GA 6194 604 -632
ATG
652c 5'-CCW AYA GTIYKI CCICCYTCYCTI 619d 1482-1508
ATA
664 5'-AAY ATG ATIACI GGIGCIGCICAR GA 619d 604 -632
ATG
561c 5'-ACI GTI CGGCCR CCCTCACGGAT 6194 1483-1505
543 5'-ATC TTA GTAGTT TCTGCTGCTGA 607 8- 30
660c 5'-GTA GAA TTGAGG ACGGTAGTTAG 607 678 -700
3S 658 5'-GAT YTA GTCGAT GATGAAGAATT 621 116 -138
659c 5'-GCT TTT TGIGTT TCWGGTTTRAT 621 443 -465
658 5'-GAT YTA GTCGAT GATGAAGAATT 621 116 -138
661c 5'-GTA GAA YTGTGG WCGATARTTRT 621 678 -700
558 5'-TCI TTY AARTAY GCITGGGT 665d 157 -176
559c 5'-CCG ACR GCRAYI GTYTGICKCAT 6654 1279-1301
813 5'-AAT CYG TYGAAA TGCAYCACGA 6654 687 -708
4S 559c 5'-CCG ACR GCRAYI GTYTGICKCAT 6654 1279-1301
a Thesesequences aligned to e
were derive corresponding
th primer.
b The ucleotide refer to theB. distasonis tuf sequence
n positions fragment
SO (SEQ ID NO. 8).
c Thesesequences fromthecomplementary d of the of
are DNA sequence the
stran
origi nating fragmentgiven the Sequence
in Listing.
d Sequences
from
databases.
132
CA 02307010 2000-OS-19
Annex I: Specific and ubiquitous primers for nucleic acid
amplification (tuf sequences) (continued).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Sequencing (continued)
primers
558 5'-TCI TTY AARTAYGCI TGGGT 665a 157- 176
815b 5'-TGG TGC ATYTCKACR GACTT 665a 686- 705
560 5'-GAY TTC ATYAARAAY ATGATYAC 665a 289- 311
559b 5'-CCG ACR GCRAYIGTY TGICKCAT 665a 1279- 1301
653 5'-GAY TTC ATIAARAAY ATGAT 665a 289- 308
559b 5'-CCG ACR GCRAYIGTY TGICKCAT 665a 1279- 1301
558 5'-TCI TTY AARTAYGCI TGGGT 665a 157- 176
655b 5'-CCR ATA CCICMRATY TTGTA 665a 754- 773
654 5'-TAC AAR ATYKGIGGT ATYGG 665a 754- 773
559b 5'-CCG ACR GCRAYIGTY TGICKCAT 665a 1279- 1301
696 5'-ATI GGI CAYRTIGAY CAYGGIAAR AC 698a 52 -77
697b 5'-CCI ACI GTICKICCR CCYTCRCG 698a 1132 -1154
911 5'-GAC GGM KKCATGCCG CARAC 853 22 -41
914b 5'-GAA RAG CTGCGGRCG RTAGTG 853 700 -720
912 5'-GAC GGC GKCATGCCG CARAC 846 20 -39
914b 5'-GAA RAG CTGCGGRCG RTAGTG 846 692 -712
913 5'-GAC GGY SYCATGCCK CAGAC 843 251 -270
915b 5'-AAA CGC CTGAGGRCG GTAGTT 843 905 -925
916 5'-GCC GAG CTGGCCGGC TTCAG 846 422 -441
561b 5'-ACI GTI CGGCCRCCC TCACGGAT 619a 1483 -1505
664 5'-AAY ATG ATIACIGGI GCIGCICAR ATG 619a 604 -632
GA
917b 5'-TCG TGC TACCCGTYG CCGCCAT 846 593 -614
a Sequences from databases.
b These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
133
CA 02307010 2000-OS-19
Annex I: Specific ubiquitous for nucleic acid
and primers
amplification (tuf
sequences)
(continued).
OriginatingDNA
fragment
SEQ ID NO. Nucleotide sequence SEQ ID Nucleotide
N0. posi tion
Seguencing (continued)
primers
1221 5'-GAY ACI CCIGGI GTI GAY TT 1230a 292 -314
CAY
1226b 5'-GTI RMR TAICCR ATY TC 1230a 2014 -2033
AAC
1222 5'-ATY GAY ACICCI CAY GTI GAY 1230a 289 -314
GGI TT
1223b 5'-AYI TCI ARRTGI TCR CCC ATI 1230a 1408 -1433
ARY CC
1224 5'-CCI GYI HTIYTI CCI ATI ATG 1230a 1858 -1881
GAR
1225b 5'-TAI CCR AACATY SMI ARI GGI 1230a 2002 -2027
TCI AC
1227 5'-GTI CCI YTIKCI ATG TTY GGI 1230a 2002 -2027
GAR TA
1229b 5'-TCC ATY TGIGCI CCI GTI ATC 698a 4- 29
GCI AT
1228 5'-GTI CCI YTIKCI ATG TTY GGI GC 1230a 2002 -2030
GAR TAY
2$ 1229b 5'-TCC ATY TGIGCI CCI GTI ATC 698a 4- 29
GCI AT
a Sequences
from databases.
b These sequences thecomplementary d of the of
are from DNA sequence the
stran
originating fragment
given in
the Sequence
Listing.
134
CA 02307010 2000-OS-19
Annex II: Specific and ubiquitous primers for nucleic acid
amplification (atpD seguences).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Bacterial species: Streptococcus
agalactiae
627 5'-ATT GTCTAT AAAAAT GGCGATAAG TC 379-383a 42- 67b
625c 5'-CGT TGAAGA CACGAC CCAAAGTAT CC 379-383a 206- 231b
628 5'-AAA ATGGCG ATAAGT CACAA.AAAG TA 379-383a 52- 77b
625c 5'-CGT TGAAGA CACGAC CCAAAGTAT CC 379-383a 206- 231b
627 5'-ATT GTCTAT AAAAAT GGCGATAAG TC 379-383a 42- 67b
626c 5'-TAC CACCTT TTAAGT AAGGTGCTA AT 379-383a 371- 396b
628 5'-AAA ATGGCG ATAAGT CACAAAAAG TA 379-383a 52- 77b
626c 5'-TAC CACCTT TTAAGT AAGGTGCTA AT 379-383a 371- 396b
Bacterial genus: Caadida
sp.
634 5'-AAC ACYGTC AGRRCI ATTGCYATG GA 460-472, 101- 1264
474-478a
635c 5'-AAA CCRGTI ARRGCR ACTCTIGCT CT 460-472, 617- 6424
474-478a
a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the S. agalactiae atpD sequence fragment
(SEQ ID N0. 380).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
d The nucleotide positions refer to the C. albicans atpD sequence fragment
(SEQ
ID N0. 460).
135
CA 02307010 2000-OS-19
Annex II: Specific and ubiquitous primers for nucleic acid
amplification (atpD sequences) (continued).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Universal primers
562 5'-CAR ATG RAY CCI CCI GGI GYI MGI 243,244,262, 528-557b
GAR ATG
264,280,284,
291,297,309,
311,315,317,
324,329,332,
334-336,339,
342,343,351,
356,357,364-
366,370,375,
379,393a
563c 5'-GGY TGR TAI ACI GCI GAI GGC AT 243,244,262, 687-712b
CCI
264,280,284,
291,297,309,
311,315,317,
324,329,332,
334-336,339,
342,343,351,
356,357,364-
366,370,375,
379,393a
564 5'-TAY GGI CAR AAY GAR CCI CCI GGI 243,244,262, 522 -550b
ATG AA
264,280,284,
291,297,309,
311,315,317,
324,329,332,
334-336,339,
342,343,351,
356,357,364-
366,370,375,
379,393a
565c 5'-GGY TGR TAI ACI GCI GAI GGD AT 243,244,262, 687 -712b
CCI
264,280,284,
291,297,309,
311,315,317,
324,329,332,
334-336,339,
342,343,351,
356,357,364-
366,370,375,
379,393a
a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the K. pneumoniae atpD sequence fragment
(SEQ ID N0. 317).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
136
CA 02307010 2000-OS-19
Annex II: Specific and ubiquitous primers for nucleic acid
amplification (atpD seguences) (continued).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Universal primers
(continued)
l0
640 5'-TCC ATG GTITWYGGI ATG AA 248,284,315, 513-535b
CAR
317,343,357,
366,370,379,393a
641c 5'-TGA TAA CCWACIGCI GGC ATA 248,284,315, 684-709b
GAI CG
15 317,343,357,
366,370,379,393a
642 5'-GGC GTI GGIGARCGI CGT GA 248,284,315, 438-460b
ACI
317,343,357,
20 366,370,379,393a
641c 5'-TGA TAA CCWACIGCI GGC ATA 248,284,315, 684-709b
GAI CG
317,343,357,
366,370,379,393a
25 Sequencing
primers
566 5'-TTY GGI GGIGCIGGI GTIGGI AARAC 6694 445- 470
567c 5'-TCR TCI GCIGGIACR TAIAYI GCYTG 669d 883- 908
3~ 566 5'-TTY GGI GGIGCIGGI GTIGGI AARAC 6694 445 -470
814 5'-GCI GGC ACGTACACI GCCTG 6664 901 -920
568 5'-RTI ATI GGIGCIGTI RTIGAY GT 669d 25 -47
5670 5'-TCR TCI GCIGGIACR TAIAYI GCYTG 669d 883 -908
35
570 5'-RTI RYI GGICCIGTI RTIGAY GT 672d 31 -53
567c 5'-TCR TCI GCIGGIACR TAIAYI GCYTG 669d 883 -908
572 5'-RTI RTI GGISCIGTI RTIGA 6694 25 -44
4~ 567c 5'-TCR TCI GCIGGIACR TAIAYI GCYTG 669d 883 -908
569 5'-RTI RTI GGISCIGTI RTIGAT AT 6714 31 -53
567c 5'-TCR TCI GCIGGIACR TAIAYI GCYTG 669d 883 -908
45 571 5'-RTI RTI GGICCIGTI RTIGAT GT 670d 31 -53
567c 5'-TCR TCI GCIGGIACR TAIAYI GCYTG 669d 883 -908
a These sequences were aligned to derive the corresponding primer.
$Q b The nucleotide positions refer to the K. pneumoniae atpD sequence
fragment
(SEQ ID NO. 317).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
d Sequences from databases.
137
CA 02307010 2000-OS-19
Annex II: Specific and ubiquitous primers for nucleic acid
ampllflcatlOn (atpD sequences) (continued).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Sequencing (continued)
primers
700 5'-TIR TIG AYGTCGART TCCCTCARG 669a 38- 61
567b 5'-TCR TCI GCIGGIACR TAIAYIGCY TG 669a 883- 908
568 5'-RTI ATI GGIGCIGTI RTIGAYGT 669a 25- 47
573b 5'-CCI CCI ACCATRTAR AAIGC 666a 1465- 1484
574 5'-ATI GCI ATGGAYGGI ACIGARGG 666a 283- 305
573b 5'-CCI CCI ACCATRTAR AAIGC 666a 1465- 1484
574 5'-ATI GCI ATGGAYGGI ACIGARGG 666a 283- 305
708b 5'-TCR TCC ATICCIARI ATIGCIATI AT 666a 1258- 1283
681 5'-GGI SSI TTYGGIISI GGIAARAC 685 694- 716
682b 5'-GTI ACI GGYTCYTCR AARTTICCI CC 686 1177- 1202
681 5'-GGI SSI TTYGGIISI GGIAARAC 685 694 -716
683b 5'-GTI ACI GGITCISWI AWRTCICCI CC 685 1180 -1205
681 5'-GGI SSI TTYGGIISI GGIAARAC 685 694 -716
699 5'-GTI ACI GGYTCYTYR ARRTTICCI CC 686 1177 -1202
681 5'-GGI SSI TTYGGIISI GGIAARAC 685 694 -716
812b 5'-GTI ACI GGITCYTYR ARRTTICCI CC 685 1180 -1205
1213 5'-AAR GGI GGIACIGCI GCIATHCCI GG 714a 697 -722
1212b 5'-CCI CCI RGIGGIGAI ACIGCWCC 714a 1189 -1211
1203 5'-GGI GAR MGIGGIAAY GARATG 709a 724 -744
1207b 5'-CCI TCI TCWCCIGGC ATYTC 709a 985- 1004
1204 5'-GCI AAY AACITCIWM YATGCC 709a 822 -842
1206b 5'-CKI SRI GTIGARTCI GCCA 709a 926 -944
4$ 1205 5'-AAY ACI TCIAWYATG CCIGT 709a 826 -845
1207b 5'-CCI TCI TCWCCIGGC ATYTC 709a 985- 1004
a Sequences from databases.
b These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
138
CA 02307010 2000-OS-19
Annex III: Internal probes for nucleic acid hybridization and
specific detection of tuf seguences.
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Bacterial species: Candida albicans
577 5'-CAT GATTGA ACC ATC CA 407-411a 406-425b
CAC
Bacterial species: Candida dubliniensis
578 5'-CAT GATTGA AGC TTC CA 412,414-415a418-437c
CAC
Bacterial species: Enterococcus faecalis
580 5'-GCT AAACCA GCT ACA ACT CCA 62-63,607a 584-6084
ATC C
603 5'-GGT ATTAAA GAC GAA TC 62-63,607a 440-4594
ACA
1174 5'-GAA CGTGGT GAA GTT 62-63,607a 398-4154
CGC
Bacterial species: Enterococcus faecium
602 5'-AAG TTGAAG TTG TTG TT 64,608a 426-445e
GTA
Bacterial s pecies: Enterococcus gallinarum
604 5'-GGT GATGAA GTA GAA GT 66,609a 419-438f
ATC
Bacterial species: Escherichia coli
579 5'-GAA GGCCGT GCT GGT AA 78 503 -522
GAG
a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the C. albicans tuf(EF-1) sequence
fragment
(SEQ ID NO. 408).
c The nucleotide positions refer to the C. dubliniensis tuf(EF-1) sequence
fragment (SEQ ID N0. 414).
d The nucleotide positions refer to the E. faecalis tuf sequence fragment (SEQ
ID NO. 607).
a The nucleotide positions refer to the E. faecium tuf sequence fragment (SEQ
ID N0. 608>.
f The nucleotide positions refer to the E. gallinarum tuf sequence fragment
(SEQ ID N0. 609).
139
CA 02307010 2000-OS-19
Annex III: Internal probes for nucleic acid hybridization and
specific detection of tuf seguences (continued).
Originating DNA fragment
SEQ ID NO. Nucleotide sequence SEQ ID Nucleotide
NO. position
Bacterial species : HSelilO~tll.lus influenzae
581 5'-ACA TCGGTG CAT TAT TAC GTG G 610a 551- 572b
Bacterial species: Staphylococcus aureus
584 5'-ACA TGACAC ATC TAA AAC AA 176- 180c 369 -3884
585 5'-ACC ACATAC TGA ATT CAA AG 176- 180c 525 -544d
586 5'-CAG AAGTAT ACG TAT TAT CA 176- 180c 545 -564d
587 5'-CGT ATTATC AAA AGA CGA AG 176- 180c 555 -5744
588 5'-TCT TCTCAA ACT ATC GTC CA 176 -180c593 -612d
Bacterial species: Staphylococcus epidermid3.s
589 5'-GCA CGA TTCTAA AA 185, 611c 445- 464e
AAC AAC
590 5'-TAT ACGTAT TATCTA AT 185, 611c 627- 646e
AAG
591 5'-TCC TGGTTC TATTAC AC 185, 611c 586- 605e
ACC
592 5'-CAA AGCTGA AGTATA AT 185, 611c 616- 635e
CGT
593 5'-TTC ACTAAC TATCGC CA 185, 611c 671- 690e
CCA
Bacterial species: Staphylococcus haemolyticus
594 5'-ATT GGT ATC CAT GAC ACT TC 186,188-190c 437-456f
595 5'-TTA AAG CAG ACG TAT ACG TT 186,188-190c 615-634f
Bacterial species: Staphylococcus hominis
596 5'-GAA ATT ATT GGT ATC AAA GA 191,193-196c 431-4508
597 5'-ATT GGT ATC AAA GAA ACT TC 191,193-196c 437-4568
598 5'-AAT TAC ACC TCA CAC AAA AT 191,193-196c 595-6148
a Sequences from databases.
b The nucleotide positions refer to the H. influenzae tuf sequence fragment
(SEQ ID N0. 610).
c These sequences were aligned to derive the corresponding probe.
d The nucleotide positions refer to the S. aureus tuf sequence fragment (SEQ
ID
N0. 179).
a The nucleotide positions refer to the S. epidermidis tuf sequence fragment
(SEQ ID N0. 611).
$0 f The nucleotide positions refer to the S. haemolyticus tuf sequence
fragment
(SEQ ID N0. 186).
g The nucleotide positions refer to the S. hominis tuf sequence fragment (SEQ
ID N0. 191).
140
CA 02307010 2000-OS-19
Annex III: Internal probes for nucleic acid hybridization and
specific detection of tuf sequences (continued).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
NO. position
Bacterial species: Staphylococcus saprophyticus
599 5'-CGG TGA AGA CGA CA 198- 200a 406 -425b
AAT AAT
600 5'-ATG CAA GAA TCA AA 198- 200a 431 -450b
GAA AGC
601 5'-GTT TCA CGT GAT CA 198- 200a 536 -555b
GAT GTA
695 5'-GTT TCA CGT GAC CA 198- 200a 563 -582b
GAT GTA
Bacterial species: Streptococcus agalactiae
582c 5'-TTT CAA CTT CGT CGT TGA CAC GAA CAG T 207-210a 404-431d
583c 5'-CAA CTG CTT TTT GGA TAT CTT CTT TAA TAC CAA CG 207-210a 433-467d
1199 5'-GTA TTA AAG AAG ATA TCC AAA AAG C 207-210a 438-462d
Bacterial species: Streptococcus pneumoniae
1201 5'-TCA AAG AAG AAA CTA AAA AAG CTG T 971,977, 513-537e
979,986a
Bacterial species: Streptococcus pyogenes
1200 5'-TCA AAG AAG AAA CTA AAA AAG CTG T 1002 473-497
Bacterial group: Enterococcus casseliflavus-flavesceas-
gallinarum group
620 5'-ATT GGTGCA TTG CTA 58,65,66a 527 -544f
CGT
1122 5'-TGG TGCATT GCT ACG 58,65,66a 529 -546f
TGG
Bacterial genus: Staphylococcus
sp.
605 5'-GAA ATGTTC CGT AAA TT 176-203a 403 -4228
TTA
606 5'-ATT AGACTA CGC TGA TG 176-203a 420 -4398
AGC
1175 5'-GTT ACTGGT GTA GAA TTC 176-203a 391 -4118
ATG
1176 5'-TAC TGGTGT AGA AAT C 176-203a 393 -4118
GTT
a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the S. saprophyticus tuf sequence fragment
(SEQ ID N0. 198).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
d The nucleotide positions refer to the S. agalactiae tuf sequence fragment
(SEQ ID N0. 209).
a The nucleotide positions refer to the S. pneumoniae tuf sequence fragment
(SEQ ID N0. 986).
f The nucleotide positions refer to the E. flavescens tuf sequence fragment
(SEQ ID NO. 65).
g The nucleotide positions refer to the S. aureus tuf sequence fragment (SEQ
IE
NO. 179).
141
CA 02307010 2000-OS-19
Annex III: Internal probes for nucleic acid hybridization and
specific detection of tuf sequences.
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Bacterial genus: StreBtococcus sp.
1202 5'-GTG TTG AAA TGT TCC GTA AAC A 206-231,971, 466-487b
977,979,982-986a
funaal species: Candida alb3cans
1156 5'-GTT GAA ATG CAT CAC GAA CAA TT 407-412,624a 680-702c
fungal species: Caadida albicans and tropicalis
1160 5'-CGT TTC TGT TAA AGA AAT TAG AAG 407-412, 748-771c
429,624a
funclalspecies: Candida dublinieasis
1166 5'-ACG TTAAGA ATG TTT CTG TCA A 414-415a 750- 7714
1168 5'-GAA CAATTG GTT GAA GGT GT 414-415a 707- 7264
funaal species: Candida glabrata
1158 5'-AAG AGGTAA TGT CTG TGG T 417 781 -799
1159 5'-TGA AGGTTT GCC AGG TGA 417 718 -735
fungal species: Csndids krusei
1161 5'-TCC AGGTGA TAA CGT TGG 422 720 -737
func~~alspecies: Csndida lusitanise and guillermondii
1162 5'-CAA GTCCGT GGA AAT GCA 418,424a 682- 699e
funaal s gecies: Candids paraps3losis
1157 5'-AAG AACGTT TCA GTT AAG GAA AT 426 749 -771
funaal s pecies: Candida zeylanoides
1165 5'-GGT TTCAAC GTG AAG AAC 432 713 -730
a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the S. pneumoniae tuf sequence fragment
(SEQ ID NO.
986).
c The nucleotide positions refer to the C. albicans tuf(EF-1) sequence
fragment (SEQ ID
No. 408).
d The nucleotide positions refer to the C. dubliniensis tuf(EF-1) sequence
fragment (SEQ
ID NO. 414).
a The nucleotide positions refer to the,C. lusitaniae tuf(EF-1) sequence
fragment (SEQ
ID NO. 424).
142
CA 02307010 2000-OS-19
Annex III: Internal probes for nucleic acid hybridization and
specific detection of tuf sequences (continued).
Originating UtvH =ragmen
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
1~
funcsal genus: Ca ndida sp.
1163 5'-GTT GGT AAC GTT AAG 407-412,414- 728- 748b
TTC AAC
415,417,418,
1$ 422,429a
1164 5'-GGT TTC GTC AAG AAC 413,416,420, 740- 757c
AAC
421,424,425,
426,428,431a
1167 5'-GTT GGT AAC GT 406-426, 428- 728- 741b
TTC
20 432, 624a
These sequenceswere alignedderive the corresponding
to primer.
The nucleotide positions to the C. albicanstuf(EF-1) sequence (SEQ
refer fragment ID
25 NO. 408).
c The nucleotidepositions to the C. lusitaniaetuf(EF-1) sequence
refer fragment (SEQ
ID NO. 424).
143
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CA 02307010 2000-OS-19
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CA 02307010 2000-OS-19
Annex X=II: Strategy for the selection of the
Staphylococcus genus-specific hybridization
probe from tuf sequences.
S
400 425 SEQ ID NO.: Accession
#:
S. aureus G TTGAAATGTTCCGTAAATTATTAGA 179 -
S. aureus G TTGAAATGTTCCGTAAATTATTAGA 176 -
S. aureus G TTGAAATGTTCCGTAAATTATTAGA 177 -
S. aureus G TTGAAATGTTCCGTAAATTATTAGA 178 -
S. aureus aureus G TTGAAATGTTCCGTAAATTATTAGA 180 -
S. auricularis G TAGAAATGTTCCGTAAATTATTAGA 181 -
1S S. capitis capitis G TAGAAATGTTCCGTAAATTATTAGA 182 -
M. caseolyticus G TAGAAATGTTCCGTAAATTATTAGA 183 -
S. cohnii G TAGAAATGTTCCGTAAATTATTAGA 184 -
S. epidermidis G TAGAAATGTTCCGTAAATTATTAGA 185 -
S. haemolyticus G TAGAAATGTTCCGTAAATTATTAGA 186 -
S. haemolyticus G TAGAAATGTTCCGTAAATTATTAGA 189 -
S. haemolyticus G TAGAAATGTTCCGTAAATTATTAGA 190 -
S. haemolyticus G TAGAAATGTTCCGTAAATTATTAGA 188 -
S. hominis G TAGAAATGTTCCGTAAATTATTAGA 196 -
S. hominis G TAGAAATGTTCCGTAAATTATTAGA 194 -
ZS S. hominis hominis G TAGAA.ATGTTCCGTAAATTATTAGA 191 -
S. hominis G TAGAAATGTTCCGTAAATTATTAGA 193 -
S. hominis G TAGAAATGTTCCGTAAATTATTAGA 195 -
S. lugdunensis G TAGAAATGTTCCGTAAATTATTAGA 197 -
S. saprophyticus G TAGAAATGTTCCGTAAATTATTAGA 198 -
S. saprophyticus G TAGAAATGTTCCGTAAATTATTAGA 200 -
S. saprophyticus G TAGAAATGTTCCGTAAATTATTAGA 199 -
S. sciuri sciuri G TTGAAATGTTCCGTAAATTATTAGA 201 -
S. warneri G TAGAAATGTTCCGTAAgTTATTAGA 187 -
S. warneri G TAGAAATGTTCCGTAAgTTATTAGA 192 -
3S S. warneri G TAGAAATGTTCCGTAAgTTATTAGA 202 -
S. warneri G TAGAAATGTTCCGTAAgTTATTAGA 203 -
B. subtilis G TTGAAATGTTCCGTAAgcTtcTTGA - Z99104
E. Coli G TTGAAATGTTCCGcAAAcTgcTGGA 78 -
L. monocytogenes G TAGAAATGTTCCGTAAATTAcTAGA 138a -
40
Selected sequence for
genus-specific hybridi-
zation probe GAAATGTT CCGTAAATTA TT 605
4S
The sequence numbering refers to the Staphylococcus aureus tuf gene fragment
(SEQ ID N0. 179). Nucleotides in capitals are identical to the selected
sequence
or match that sequence. Mismatches are indicated by lower-case letters.
SO a The SEQ ID N0. refers to previous patent application W098/20157.
1SS
CA 02307010 2000-OS-19
Annex XIV: Strategy for the selection of Staphylococcus
saprophyt3cus-specific and of Staphylococcus
haemolyticus-specific hybridization probes
from tuf seguences.
SEQ
ID
1~ 339 383 NO.:
S. aureus AG TtGGTGAAGAAyTtGAAATCATcGGTtTaCATGACACaTCTAA 179
S. aureus AG TtGGTGAAGAAgTtGAAATCATcGGTtTaCATGACACaTCTAA 176
S. aureus AG TtGGTGAAGAAgTtGAAATCATcGGTtTaCATGACACaTCTAA 177
S. aureus AG TtGGTGAAGAAgTtGAAATCATCGGTtTaCATGACACaTCTAA 178
S. aureus aureus AG TtGGTGAAGAAgTtGAAATCATcGGTtTaCATGACACaTCTAA 180
S. auricularis AG TCGGTGAAGAAgTtGAAATCATcGGTATgaAaGACggTTCAAA 181
S. capitis capitis AG TtGGTGAAGAAgTtGAAATCATcGGTATCCAcGAs.ACTTCTAA 182
M. caseolyticus AG TtGGTGAAGAAgTtGAAATCATTGGTtTaacTGAagaacCAAA 183
S. cohnii AG TCGGTGAAGAAgTtGAAATCATcGGTATgCAaGAagaTTCCAA 184
ZO S. epidermidis AG TtGGTGAAGAAgTtGAAATCATcGGTATgCAcGAaACTTCTAA 185
S. haemolyticus AG TtGGTGAAGAAgTtGAAATCATTGGTATCCATGACACTTCTAA 186
S. haemolyticus AG TtGGTGAAGAAgTtGAAATCATTGGTATCCATGACACTTCTAA 189
S. haemolyticus AG TtGGTGAAGAAgTtGAAATCATTGGTATCCATGACACTTCTAA 190
S. haemolyticus AG TtGGTGAAGAAgTtGAAATtATTGGTATCaAaGAsACTTCTAA 188
ZS S. hominis AG TtGGTGAAGAAgTtGAAATtATTGGTATCaAaGAsACTTCTAA 194
S. hominis hominis AG TtGGTGAAGAAgTtGAAATtATTGGTATCaAaGAaACTTCTAA 191
S. hominis AG TtGGTGAAGAAgTtGAAATtATTGGTATCaAaGAaACTTCTAA 193
S. hominis AG TtGGTGAAGAAgTtGAAATtATTGGTATCaAaGAaACTTCTAA 195
S. hominis AG TtGGTGAAGAAgTtGAAATtATTGGTATCaAaGAtACTTCTAA 196
S. lugdunensis AG TCGGTGAAGAAgTtGAAATtATTGGTATCCAcGAtACTaCTAA 197
S. saprophyticus AG TCGGTGAAGAAATCGAAATCATcGGTATgCAaGAagaaTCCAA 198
S. saprophyticus AG TCGGTGAAGAAATCGAAATCATCGGTATgCAaGAagaaTCCAA 200
S. saprophyticus AG TCGGTGAAGAAATCGAAATCATcGGTATgCAaGAagaaTCCAA 199
S. sciuri sciuri TG TtGGTGAAGAAgTtGAAATCATcGGTtTaacTGAagaaTCTAA 201
3S S. warneri AG TtGGTGAAGAAgTtGAAATCATcGGTtTaCATGACACTTCTAA 187
S. warneri AG TtGGTGAAGAAgTtGAAATCATcGGTtTaCATGACACTTCTAA 192
S. warneri AG TtGGTGAAGAAgTtGAAATCATcGGTtTaCATGACACTTCTAA 202
S. warneri AG TtGGTGAAGAAgTtGAAATCATcGGTtTaCATGACACTTCTAA 203
B. subtilis AG TCGGTGACGAAgTtGAAATCATCGGTCTtCAaGAagagagAAA -a
4O E. coli AG TtGGTGAAGAAgTtGAAATCgTTGGTATCaAaGAgACTcaGAA 78
L. monocytogenes AG TtGGTGAcGAAgTaGAAgTtATCGGTATCgAaGAagaaagAAA 138b
Selected sequences for
species-specific
45 hybridization probes CGGTGAAGA AATCGAAATC A 599
ATTGGTATCC ATGACACTTC 594
The sequence numbering refers to the Staphylococcus aureus tuf gene fragment
(SEQ ID N0. 179). Nucleotides in capitals are identical to the selected
50 sequences or match those sequences. Mismatches are indicated by lower-case
letters.
This sequence was obtained from Genbank accession #Z99104.
The SEQ ID NO. refers to previous patent application W098/20157.
156
CA 02307010 2000-OS-19
Annex Xv: Strategy for the selection of Staphylococcus
aureus-specific and of Staphylococcus
epidermidis-specific hybridization probes from
tuf seguences.
S
SEQ
ID
521 547 592 617 NO.:
IO S. aureus TACACCACATACTGAATTC AAAGCAG...TTCTTCtCa
AACTATCGtCCACAATT179
S. aureus TACACCACATACTGAATTC AAAGCAG...TTCTTCtC --- 178
S. aureus TACACCACATACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtCCACAATT176
S. aureus TACACCACATACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtCCACAATT177
S. aureus aureus TACACCACATACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtCCACAATT180
IS S. auricularis TACACCACAcACTaAATTC ActGCAG...TTCTTCtCT
AACTACCGtCCACAATT181
S. capitis capitisCACACCACAcACTaAATTC AAAGCGG...TTCTTCAgT AACTACCGCCCACAATT182
M. caseolyticus TACtCCACATACTaAATTC AAAGCTG...TTCTTCACT AACTACCGCCCtCAGTT183
S. cohnii TACACCACAcACaeACTTt AAAGCGG...TTCTTCAgT AACTATCGCCCACAATT184
S. epidermidis TACACCACAcACaaAATTC AAAGCTG...TTCTTCACT AACTATCGCCCACAATT185
ZO S. haemolyticus CACACCtCAcACaaAATTt AAAGCAG...TTCTTCACa
AACTATCGtCCACAATT186
S. haemolyticus CACACCtCAcACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtCCACAATT189
S. haemolyticus CACACCtCAcACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtCCACAATT190
S. haemolyticus TACACCtCAcACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtCCACAATT188
S. hominis CACACCtCACACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtCCACAATT195
ZS S. hominis TACACCtCAcACasAATTC AAAGCAG...TTCTTCACT
AACTATCGtCCACAATT196
S. hominis hominisTACACCtCAcACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtCCACAATT191
S. hominis TACACCtCAcACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtCCACAATT193
S. hominis TACACCtCAcACasAATTC AAAGCAG...TTCTTCtCT AACTATCGtCCACAATT194
S. lugdunensis TACACCtCAcACTaAATTt AAAGCTG...TTCTTCtCa AACTACCGCCCACAATT197
3O S. saprophyticus TACACCACATACaaAATTC AAAGCGG...TTCTTCACT
AACTACCGCCCACAATT198
S. saprophyticus TACACCACATACaaAATTC AAAGCGG...TTCTTCACT AACTACCGCCCACAATT199
S. saprophyticus TACACCACATACaaAATTC AAAGCGG...TTCTTCACT AACTACCGCCCACAATT200
S. sciuri sciuri CACACCtCAcACTaAATTC AAAGCTG...TTCTTCACa AACTAcCGCCCACAATT201
S. warneri TACACCACATACaaAATTC AAAGCGG...-- --- 192
35 S. warneri TACACCACATACaaAATTC AAAGCGG...TTCTTCAQT
AACTAcCGCCCACAATT187
S. warneri TACACCACATACaaAATTC AAAGCGG...TTCTTCAgT AACTACCGCCCACAATT202
S. warneri TACACCACATACaaAATTC AAAGCGG...TTCTTCAgT AACTACCGCCCACAATT203
B. subti.li.s CACtCCACAcAgcaAATTC AAAGCTG...TTCTTCtCT AACTACCGtCCtCAGTT-a
E. coli CAAgCCgCAcACcaAgTTC gAAtCTG...TTCTTCAaa ggCTAcCGtCCgCAGTT78
4O L. monocytogenes TACtCCACAcACTaACTTC AAAGCTG...TTCTTCAac
AACTACCGCCCACAATT138b
Selected
sequences
for
species-specific
hybridization
4S probes ACCACA TACTGAATTC AAAG 585
TTCACT AACTATCGCCCACA 593
The sequence numbering refers to the Staphylococcus aureus tuf gene fragment
(SEQ ID N0.
179). Nucleotides in capitals are identical to the selected sequences or match
those
SO sequences. Mismatches are indicated by lower-case letters. - indicate
incomplete
sequence data. Dots indicate gaps in the sequences displayed.
This sequence was obtained from Genbank accession #Z99104.
The SEQ ID NO. refers to previous patent application W098/20157.
1S7
CA 02307010 2000-OS-19
Annex Xv=: Strategy for the selection of the
Staphylococcus hominis-specific hybridization
probe from tuf seguences.
358 383 SEQ ID NO
S. aureus ATC ATCGGTtTac AtGAcACaTCTAA 179
S. aureus ATC ATcGGTtTac AtGAcACaTCTAA 176
S. aureus ATC ATcGGTtTac AtGAcACaTCTAA 177
S, aureus ATC ATCGGTtTac AtGAcACaTCTAA 178
5. aureus aureus ATC ATCGGTtTac AtGAcACaTCTAA 180
S. auricularis ATC ATCGGTATgA AAGAcggTTCAAA 181
S. capitis capitis ATC ATcGGTATCc AcGAAACTTCTAA 182
M. caseolyticus ATC ATTGGTtTaA ctGAAgaacCAAA 183
S. cohnii ATC ATcGGTATgc AAGAAQaTTCCAA 184
S. epidermidis ATC ATCGGTATgc AcGAAACTTCTAA 185
S. haemolyticus ATC ATTGGTATCc AtGAcACTTCTAA 186
S. haemolyticus ATC ATTGGTATCc AtGAcACTTCTAA 189
S. haemolyticus ATC ATTGGTATCc AtGAcACTTCTAA 190
S. haemolyticus ATT ATTGGTATCA AAGAAACTTCTAA 188
S. hominis ATT ATTGGTATCA AAGAtACTTCTAA 196
S. hominis ATT ATTGGTATCA AAGAAACTTCTAA 194
S. hominis hominis ATT ATTGGTATCA AAGAAACTTCTAA 191
S. hominis ATT ATTGGTATCA AAGAAACTTCTAA 193
S. hominis ATT ATTGGTATCA AAGAAACTTCTAA 195
S. lugdunensis ATT ATTGGTATCc AcGAtACTaCTAA 197
S. saprophyticus ATC ATCGGTATgc AAGAAgaaTCCAA 198
S. saprophyticus ATC ATCGGTATgc AAGAAgaaTCCAA 200
S. saprophyticus ATC ATcGGTATgc AAGAAgaaTCCAA 199
S. sciuri sciuri ATC ATcGGTtTaA ctGAAgaaTCTAA 201
S. warneri ATC ATcGGTtTac AtGAcACTTCTAA 187
S. warneri ATC ATCGGTtTac AtGACACTTCTAA 192
S. warneri ATC ATCGGTtTac AtGAcACTTCTAA 202
S. warneri ATC ATCGGTtTac AtGAcACTTCTAA 203
B. subtilis ATC ATCGGTCTtC AAGAAgagagAAA -a
E. coli ATC gTTGGTATCA AAGAgACTcaGAA 78
L. monocytogenes GTT ATcGGTATCg AAGAAgaaagAAA 138b
Se lected sequence
for
species-specific
hybridization ATTGGTATCA AAGAAACTTC 597
probe
50
The sequence numbering refers to the Staphylococcus aureus tuf gene fragment
(SEQ ID NO. 179). Nucleotides in capitals are identical to the selected
sequences or match those sequences. Mismatches are indicated by lower-case
letters. Dots indicate gaps in the sequences displayed.
This sequence was obtained from Genbank accession #Z99104.
The SEQ ID NO. refers to previous patent application W098/20157.
158
CA 02307010 2000-OS-19
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CA 02307010 2000-OS-19
Annex XXI: Specific and ubiguitous primers for nucleic acid
amplification (recA seguences).
OriginatingDNA fragment
SEQ ID NO. Nucleotide sequence SEQ ID Nucleotide
N0. position
Universal primers (recA)
919 5'-GGI CCI GAR TCI TMI GGI AC 918a 437-459
AAR
920b 5'-TCI CCV ATI TCI CCI TCI TC 918a 701-723
AIY
921 5'-TIY RTI GAY GCI GAR CAI 918a 515-534
GC
922b 5'-TAR AAY TTI ARI GCI YKI CC 918a 872-894
CCI
Universal primers (rad51)
935 5'-GGI AAR WSI CAR YTI TGY AC 939a 568-590
CAY
936b 5'-TCI SIY TCI GGI ARR CAI 939a 1126-1145
GG
Universal primers (dmcl)
937 5'-ATI ACI GAR GYI TTY GGI TT 940a 1038-1060
GAR
938b 5'-CYI GTI GYI SWI GCR TGI 940a 1554-1573
GC
30a Sequences
from databases.
b These sequences the complementary and of the
are from DNA str sequence
of the
originating fragment
given in
the Sequence
Listing.
163
CA 02307010 2000-OS-19
Annex XXII: Specific and ubiguitous primers for nucleic acid
amplification (speA seguences).
OriginatingDNA fragment
SEQ ID NO. Nucleotide sequence SEQ ID Nucleotide
N0. position
Bacterial pecies: Streptococcus pyogenes
s
994 5'-TGG ACT AAC AAT CTC GCA AGA 993a 60-82
GG
995b 5'-ACA TTC TCG TGA GTA ACA GGG 993a 173-194
T
996 5'-ACA AAT CAT GAA GGG AAT CAT 993a 400-424
TTA G
997b 5'-CTA ATT CTT GAG CAG TTA CCA 993a 504-526
TT
998 5'-GGA GGG GTA ACA AAT CAT GAA 993a 391-413
GG
997b 5'-CTA ATT CTT GAG CAG TTA CCA 993a 504-526
TT
a Sequence
from databases.
These sequences
are from
the complementary
DNA strand
of the sequence
of the
originating fragment given in the Sequence
Listing.
164
CA 02307010 2000-OS-19
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CA 02307010 2000-OS-19
riiinBX xxxIIES~eClflC and ubiguitous primers
for nuCl~1C aCl.d
aI~11f1Catl.On
( stX SegllenC88
~ .
OriginatingDNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
NO. position
Toxin gene stx
:
l
1081 5'-ATG TCA GAG GGA TAG ATC CA 1076a 233-252
1080b 5'-TAT AGC TAC TGT CAC CAG ACA 1076a 394-418
ATG T
Toxin aene stx
:
?
1078 5'-AGT TCT GCG TTT TGT CAC TGT 1077a 546-567
C
1079b 5'-CGG AAG CAC ATT GCT GAT T 1077a 687-705
Toxin aenes stx
: and stx
l
s
1082 5'-TTG ARC RAA ATA ATT TAT ATG 1076a 287-309
TG
1083b 5'-TGA TGA TGR CAA TTC AGT AT 1076a 790-809
a Sequences
from databases.
b These sequences the complementary DNA
are from strand of the sequence
of the
originating fragment n in the Sequence Listing.
give
175
CA 02307010 2000-OS-19
Annex XXXIII: Molecular beacon internal probes for hybridization
and specific detection of toxin sequences.
Originating DNA fragment
SEQ ID N0. Nucleotide sequenceaSEQ ID Nucleotide
N0. position
1~
Toxin gene: stxl
1084 5'-CCA CGC CGC TTT GCT GAT TTT TCA CAT
GTT ACC GCG TGG 1076b 346-372
1S
Toxin gene: stxa
1085 5'-CCA CGC CAC TGT CTG AAA CTG CTC CTG
TG CGT GG 1077b 617-638
20
a Underlined nucleotides indicatethe molecular beacon's stem.
Sequences from databases.
176
CA 02307010 2000-OS-19
Annex XXXIV: Specific and ubiquitous primers for nucleic acid
amplification (van sequences).
Originating DNA fragment
SEQ ID NO. Nucleotide sequence SEQ ID Nucleotide
NO. position
Resistance gene: vanA
1086 5' -CTACTCCCGCCT TTTGGGTT 1049-1057a 513-532b
1087c 5' -CTCACAGCCCGA AACAGCCT 1049-1057a 699-718b
1086 5' -CTACTCCCGCCT TTTGGGTT 1049-1057a 513-532b
1088c 5' -TGCCGTTTCCTG TATCCGTC 1049-1057a 885-904b
1086 5' -CTACTCCCGCCT TTTGGGTT 1049-1057a 513-532b
1089c 5' -ATCCACACGGGC TAGACCTC 1049-1057a 933-952b
1090 5' -AATAGCGCGGAC GAATTGGAC 1049-1057a 629-649b
1091c 5' -AACGCGGCACTG TTTCCCAA 1049-1057a 734-753b
1090 5' -AATAGCGCGGAC GAATTGGAC 1049-1057a 629-649b
1089c 5' -ATCCACACGGGC TAGACCTC 1049-1057a 933-952b
1092 5' -TCGGCAAGACAA TATGACAGC 1049-1057a 662-682b
1088c 5' -TGCCGTTTCCTG TATCCGTC 1049-1057a 885-904b
Resistance aene: vane
1095 5' -CGATAGAAGCAG CAGGACAA 11174 473-492
1096c 5' -CTGATGGATGCG GAAGATAC 11174 611-630
Resistance gene: vanAB
1112 5' -GGCTGYGATATT CAAAGCTC 1049-1057,1117a437-456b
1113c 5' -ACCGACCTCACA GCCCGAAA 1049-1057,1117a705-724b
1112 5' -GGCTGYGATATT CAAAGCTC 1049-1057,1117a437-456b
11140 5' -TCWGAGCCTTTT TCCGGCTCG 1049-1057,1117a817-837b
1115 5' -TTTCGGGCTGTG AGGTCGGBT GHG 1049-1057,1117a705-730b
CG
1114c 5' -TCWGAGCCTTTT TCCGGCTCG 1049-1057,1117a817-837b
1116 5' -TTTCGGGCTGTG AGGTCGGBT GHG 1049-1057,1117a705-731b
CGG
1114c 5' -TCWGAGCCTTTT TCCGGCTCG 1049-1057,1117a817-837b
1112 5'-GGC TGY GAT ATT CAA AGC TC 1049-1057,1117a 437-456b
$0 1118c 5'-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057,1117a 817-840b
a These sequences were aligned to derive the corresponding primer.
The nucleotide positions refer to the vanA sequence fragment (SEQ ID NO.
1051).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
d Sequences from databases.
177
CA 02307010 2000-OS-19
Annex xxxIV: Specific and ubiquitous primers for nucleic acid
amplification sequences) (continued).
(van
Originating
DNA fragment
SEQ ID Nucleotide sequence SEQ ID
Nucleotide
N0.
N0. position
Resistance g-ene: vanAB (continued)
_
1115 5' -TTTCGGGCTGTG AGGTCG GBTGHGCG 1049-1057,1117a705-730b
1118c 5' -TTTTCWGAGCCT TTTTCC GGCTCG 1049-1057,1117a817-840b
1116 5' -TTTCGGGCTGTG AGGTCG GBTGHGCGG 1049-1057,1117a705-731b
1118c 5' -TTTTCWGAGCCT TTTTCC GGCTCG 1049-1057,1117a817-840b
1119 5' -TTTCGGGCTGTG AGGTCG GBTGHGC 1049-1057,1117a705-729b
1118c 5' -TTTTCWGAGCCT TTTTCC GGCTCG 1049-1057,1117a817-840b
1120 5' -TTTCGGGCTGTG AGGTCG GBTGHG 1049-1057,1117a705-728b
1118c 5' -TTTTCWGAGCCT TTTTCC GGCTCG 1049-1057,1117a817-840b
1121 5' -TGTTTGWATTGT CYGGYA TCCC 1049-1057,1117a408-429b
1111c 5' -CTTTTTCCGGCT CGWYTT CCTGATG 1049-1057,1117a806-830b
1112 5' -GGCTGYGATATT CAAAGC TC 1049-1057,1117a437-456b
1111c 5' -CTTTTTCCGGCT CGWYTT CCTGATG 1049-1057,1117a806-830b
1123 5' -TTTCGGGCTGTG AGGTCG GBTG 1049-1057,1117a705-726b
1111c 5' -CTTTTTCCGGCT CGWYTT CCTGATG 1049-1057,1117a806-830b
1112 5' -GGCTGYGATATT CAAAGC TC 1049-1057,1117a437-456b
1124c 5' -GATTTGRTCCAC YTCGCC RACA 1049-1057,1117a757-778b
Resistance gene: vanCl
1103 5' -ATCCCGCTATGA AAACGA TC 1058-1059a 519-5384
1104c 5' -GGATCAACACAG TAGAAC CG 1058-1059a 678-6974
Resistance gene: vanCl ,
vanC2,
vanC3
1097 5' -TCYTCAAAAGGG ATCACW AAAGTMAC 1058-1066a 607-6324
1098c 5' -TCTTCAAAATCG AAAAAG CCGTC 1058-1066a 787-8094
1099 5' -TCAAAAGGGATC ACWAAA GTMAC 1058-1066a 610-6324
1100c 5' -GTAAAKCCCGGC ATRGTR TTGATTTC 1058-1066a 976-10014
1101 5' -GACGGYTTTTTY GATTTT GAAGA 1058-1066a 787-8094
1102c 5' -AAAAARTCGATK CGAGCM AGACC 1058-1066a 922-9444
Resistance ene: vanC2-vanC3
_
1105 5' -CTCCTACGATTC TCTTGA YAAATCA 1060-1066,1140a487-511e
1106c 5' -CAACCGATCTCA ACACCG GCAAT 1060-1066,1140a690-712e
a These sequences were aligned to derive the corresponding primer.
The nucleotide positions refer to the vanA sequence fragment (SEQ ID NO.
1051).
c These sequences are from the complementary DNA strand of the sequence of the
originating fragment
given in the Sequence Listing.
d The nucleotide positions refer to the vanCl sequence fragment (SEQ ID NO.
1058).
The nucleotide positions refer to the vanC2 sequence fragment (SEQ ID NO.
1140).
178
CA 02307010 2000-OS-19
Annex XXXIYa Spec7.flC and ubiquitous primers for nucleic acid
amplification (van sequences) (continued).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Sequencing primers (vanAB)
1112 5'-GGCTGY GATATT CAA AGC TC 1139a 737- 756
1111b 5'-CTTTTT CCGGCT CGW YTT CCT GAT 1139a 1106-1130
G
Sequencing primers ( vanA, vanX,vanY)
1150 5'-TGATAA TCACAC CGC ATA CG 1141a 860- 879
1151b 5'-TGCTGT CATATT GTC TTG CC 1141a 1549-1568
1152 5'-ATAAAG ATGATA GGC CGG TG 1141a 1422-1441
1153b 5'-CTCGTA TGTCCC TAC AAT GC 1141a 2114-2133
1154 5'-GTTTGA AGCATA TAG CCT CG 1141a 2520-2539
1155b 5'-CAGTGC TTCATT AAC GTA GTC 1141a 3089-3109
Sequencing primers (vanC1)
1110 5'-ACGAGA AAGACA ACA GGA AGA CC 1138a 122- 144
1109b 5'-ACATCG TGATCG CTA AAA GGA GC 1138a 1315-1337
Seguencing primers (vanC2-vanC3)
1108 5'-GTAAGA ATCGGA AAA GCG GAA GG 1140a 1-23
1107b 5'-CTCATT TGACTT CCT CCT TTG CT 1140a 1064-1086
a Sequencesfrom
databases.
These sequences are from strand of
the the sequence
complementary of the
DNA
originating mentgiven in the Sequence ng.
frag Listi
179
CA 02307010 2000-OS-19
Annex XXXV: Internal probes for nucleic acid hybridization and
specific detection of van sequences.
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Resistance gene: vanA
1170 5'- ACG AAT TGG ACG CAA TT 1049-1057a 639-658b
ACT
15Resistance aene: vasiB
1171 5'- ACG AGG ATG TGA TTG TC 1117c 560-579
ATT
a These sequences were aligned to derive the corresponding primer.
b The nucleotide positions refer to the vanA sequence fragment (SEQ ID N0.
1051).
c Sequences from databases.
180
CA 02307010 2000-OS-19
Annex XXXVI: Specific and ubiguitous primers for nucleic acid
amplification~(pbp sequences).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Resistance aene: pbpla
1129 5'-ATG ATGACC GAM 1004-1018a 681-703b
ATG
ATG
AAA
AC
11310 5'-CAT CTGGAG CTACRT ARC CAG 1004-1018a 816-837b
T
1130 5'-GAC TATCCA AGCATG CAT TAT 1004-1018a 456-477b
G
1131 5'-CAT CTGGAG CTACRT ARC CAG 1004-1018a 816-837b
T
Se guencing (pbpla)
primers
1125 5'-ACT CACAAC TGGGAT GGA TG 11694 873 -892
1126c 5'-TTA TGGTTG TGCTGG TTG AGG 11694 2140-2160
1125 5'-ACT CACAAC TGGGAT GGA TG 11694 873 -892
1128c 5'-GAC GACYTT ATKGAT ATA CA 11694 1499-1518
1127 5'-KCA AAYGCC ATTTCA AGT AA 11694 1384-1403
1126c 5'-TTA TGGTTG TGCTGG TTG AGG 11694 2140-2160
Sequencing (pbp2b)
primers
1142 5'-GAT CCTCTA AATGAT TCT CAG G 11724 1- 25
GTG
1143c 5'-CAA TTAGCT TAGCAA TAG GTG G 11724 1481-1505
TTG
1142 5'-GAT CCTCTA AATGAT TCT CAG G 11724 1- 25
GTG
1145c 5'-AAC ATATTK GGTTGA TAG GT 11724 793-812
1144 5'-TGT YTTCCA AGGTTC AGC TC 11724 657-676
1143c 5'-CAA TTAGCT TAGCAA TAG GTG G 11724 1481-1505
TTG
Seguencing (pbp2x)
primers
1146 5'-GGG ATTACC TATGCC AAT ATG 11734 219-241
AT
1147c 5'-AGC TGTGTT AGCVCG AAC ATC 11734 1938-1961
TTG
1146 5'-GGG ATTACC TATGCC AAT ATG 11734 219-241
AT
1149c 5'-TCC YACWAT TTCTTT TTG WG 11734 1231-1250
1148 5'-GAC TTTGTT TGGCGT GAT AT 11734 711-730
11470 5'-AGC TGTGTT AGCVCG AAC ATC 11734 1938-1961
TTG
a These sequences aligned to
were derive
the
corresponding
primer.
The nucleot ide positions ref er quence fragment(SEQID
to NO
the
pbpla
se
.
1004.
c These sequences the complementary trand of the
are from DNA sequence of
s the
originating fragment theSequence Listing.
given
in
d Sequences
from databases.
181
CA 02307010 2000-OS-19
Annex XXXVII: Internal probes for nucleic acid hybridization
and speclflC detection of pbp sequences.
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Resistance gene: pbpla
1132 5' -AGTGAAAAR ATGGCT GCTGC 1004-1018a 531-550b
1133 5' -CATCAAGAA CACTGG CTAYGTAG 1004-1018a 806 -828b
1134 5' -CTAGATAGA GCTAAA ACCTTCCT 1004-1018a 417 -439b
1135 5' -CATTATGCA AACGCC ATTTCAAG 1004-1018a 471 -493b
1192 5' -GGTAAAACA GGAACC TCTAACT 1004-1018a 759 -780b
1193 5' -GGTAAGACA GGTACT TCTAACT 1004-1018a 759 -780b
1194 5' -CATTTCAAG TAATAC AACAGAATC 1004-1018a 485 -508b
1195 5' -CATTTCAAG TAACAC AACTGAATC 1004-1018a 485- 508b
1196 5' -GCCATTTCA AGTAAT ACAACAGAA 1004-1018a 483- 506b
1197 5' -CAAACGCCA TTTCAA GTAATACAAC 1004-1018a 478- 502b
1094 5' -GGTAAAACA GGTACT TCTAACTA 1004-1018a 759- 781b
1214 5' -GGTAAAACA GGTACC TCTAACTA 1004-1018a 759- 781b
2$ 1216 5' -GGTAAGACT GGTACA TCAAACTA 1004-1018a 759- 781b
1217 5' -CAAATGCCA TTTCAA GTAACACAAC 1004-1018a 478-502b
1218 5' -CAAACGCCA TTTCAA GTAACACAAC 1004-1018a 478- 502b
1219 5' -CAAATGCTA TTTCAA GTAATACAAC 1004-1018a 478- 502b
1220 5' -CAAACGCCA TTTCAA GTAATACGAC 1004-1018a 478- 502b
a These sequences were aligned to derive the corresponding primer.
The nucleotide positions refer to the pbpla sequence fragment (SEQ ID N0.
1004).
182
CA 02307010 2000-OS-19
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N ~ ~ N N N N M O ~ ~O O M O tf7 O ~O ~ O CT O N U U
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U v0 h ~ ~ ~ cN V' M .-i u'1 N ~O tf7 O r-i c-~ N 1~ N 1~ 1~ ~-I ~ N
U Lf1 01 01 a1 01 01 01 00 O M h O rl O C~ w b (0 41 U7 ~ = N
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O v7 O N 0~.,~ M
CA 02307010 2000-OS-19
Annex XXXIX: Internal probe for nucleic acid hybridization and
specific detection of mecA.
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
1~
Resistance gene: mecA
1177 5'-GCT CAA CAA GTT CCA GAT TA 1178a 1313-1332
15
a Sequence from databases.
185
CA 02307010 2000-OS-19
Annex XL: Specific and ubiquitous primers for nucleic acid
amplification (hexA seguences).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Bacterial species: Streptococcus pneumoaiae
1179 5'-ATT TGG CGGGTG ACT TT 1183a 431-450
TGA
1181b 5'-AGC AGC CTAGAT GCC GT 1183-1191c 652-671d
TTA
Sequencing
primers
1179 5'-ATT TGG CGGGTG ACT TT 1183a 431-450
TGA
1182b 5'-AAC TGC AGATCC TTT GG 1183a 1045-1064
AAG
a Sequences from databases.
b These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
c These sequences were aligned to derive the corresponding primer.
d The nucleotide positions refer to the hexA sequence fragment (SEQ ID N0.
1183).
186
CA 02307010 2000-OS-19
Annex XLI: Internal probe for nucleic acid hybridization and
specific detection of hexA sequences.
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Bacterial species: Streptococcus pneumoniae
1180a 5'-TCC ACC GTT GCC AAT CGC A 1183-1191b 629-647c
a This sequences is from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
b These sequences were aligned to derive the corresponding primer.
c The nucleotide positions refer to the hexA sequence fragment (SEQ ID N0.
1183 ) .
187
<IMG>
CA 02307010 2000-OS-19
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<IMG>
CA 02307010 2000-OS-19
Annex XLIII: Specific and ubiquitous primers for nucleic acid
amplification (pcp sequence).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
1~
Bacterial species: Streptococcus pyogenes
1211 5'-ATT CTT GTA ACA GGC TTT GAT CCC 1215a 291-314
1210b 5'-ACC AGC TTG CCC AAT ACA AAG G 1215a 473-494
a Sequences from databases.
These sequences are from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
191
CA 02307010 2000-OS-19
Annex XLIV: Specific and ubiguitous primers for nucleic acid
amplification (S. saprophyticus unknov~m gene
sequences).
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Bacterial species: Staphylococcus saprophyticus
1208 5'-TCA AAA AGT TTT CTA AAA AAT TTA C 74,1093, 169-193c
1198b
1209a 5'-ACG GGC GTC CAC AAA ATC AAT AGG A 74,1093, 355-379c
1198b
a This sequence is from the complementary DNA strand of the sequence of the
originating fragment given in the Sequence Listing.
b These sequences were aligned to derive the corresponding primer.
c The nucleotide positions refer to the S. saprophyticus unknown gene sequence
fragment (SEQ ID N0. 1198).
192
CA 02307010 2000-OS-19
S
Annex XLV: Molecular beacon internal probes for hybridization
and specific detection of antibiotic resistance
gene sequences.
OriginatingDNA fragment
SEQ ID NO. sequences SEQ ID Nucleotide
Nucleotide
N0. position
Resistance aene: mecA
1231 5' -GCG CCG AAG AAA AACCTC 1178b 1291-1315
AGC ATA AAG TGC
IS T~ TCG ~
Resistance gene: vanA
1239 5' -GCG GCA GAC TCA GAGGAG 1051 860-880
AGC CTT GCA GCT
CGC
1240 5' -GCG CGG CAA AAT ACAGCA 1051 663-688
AGC GAC ATG AAA
TCC~
CTC
2S Resistance ~: vanB
_
1241 5' -GCG GGG GAA GGA TTTGAT 1117 555-577
AGC CGA TGA TGC~
CTC
s Underlined nucleotides indicate the molecular beacon's stem.
Sequence from databases.
193
CA 02307010 2000-OS-19
Annex XLVI: Molecular beacon internal probe for hybridization
and specific detection of an unknown S. aureus gene
sequence.
Originating DNA fragment
SEQ ID N0. Nucleotide sequencea SEQ ID Nucleotide
N0. position
1~
Bacterial species: S. sureus
1232 5'-GGA ~ C~CG CGA TTT TAT TGA ATG TTG 1244 3-80
AAA 5
IS ATA ACQ GGC TCC
a Underlined nucleotides indicate the molecular beacon's stem.
194
CA 02307010 2000-OS-19
Annex XLYII: Molecular beacon internal probes for hybridization
and specific detection of tuf sequences.
$ Originating DNA fragment
SEQ ID NO. Nucleotide sequencea SEQ ID Nucleotide
N0. position
Bacterial species: Enterococcus faecalis
1236 5'-GCG CGT GGT GAA GTT CGC GTT GGT 883 370-391
AGC
GEC
TCG
Bacterial species: Enterococcus faecium
1235 5'-GCG CGA AGT TGA AGT TGT TGG TAT 64 412-437
AGC
TGC CTC CSC
TG~
2 0
Bacterial species: Staphylococcus sp. other than aureus
S.
1233 5'-GCG GTT ACT GGT GTA GAA ATG TTC 878 372-394
AGC
CGC~ CC
CTC
2 5
a Underlined nucleotides indicate the molecular beacon's stem.
195
CA 02307010 2000-OS-19
Annex XLVIII: Molecular beacon internal probes for hybridization
and specific detection of dd1 and mt1 gene
sequences.
Originating DNA fragment
SEQ ID N0. Nucleotide sequencea SEQ ID Nucleotide
N0. position
1~
Bacterial species: E. faec3um (ddl)
1237 5'-GCG CGC GAA ATC GAA GCT GTA 1242 334-359
AGC GTT
1S TTA GG_G CTC G~
Bacterial species: E, faecalis (mtl)
1238 5'-~ AGC GGC GTT AAT TTT ACC GAA 1243 631-656
GGC
GAA GAS CTC CSC
a Underlined nucleotides indicate the molecular beacon's stem.
196
CA 02307010 2000-OS-19
Annex XLIX: Internal probe for nucleic acid hybridization and
specific detection of an unknown S. aureus gene
sequence.
Originating DNA fragment
SEQ ID N0. Nucleotide sequence SEQ ID Nucleotide
N0. position
Bacterial species: Staphylococcus aureus
1234 5'-ACT AAA TAA ACG CTC ATT CG 1244 35-54
197
CA 02307010 2000-OS-19
SEQUENCE LISTING
(1)GENERAL INFORMATION:
(i)APPLICANTS:
BERGERON, Michel G. 1, 1145 des Erables, Quebec City,
Quebec, Canada, G2K 1T8
BOISSINOT, Maurice 1, 109 Jean-Bruchesi, St-Augustin-
de-Desmaures, Quebec, Canada, G3A 2N2
HULETSKY, Ann 1, 1231 Av des Pins, Sillery, Quebec,
Canada, G1S 4J3
MENARD, Christian 1, 1174 Rue du Pont, St-Lambert-de-
Levis, Quebec, Canada, GOS 2W0
OUELLETTE, Marc 1, 1035 de Ploermel, Sillery, Quebec,
Canada, G1S 3S1
PICARD, Fran~ois J. ', 1245 de la Sapiniere, Cap-Rouge,
Quebec, Canada, G1Y 1A1
ROY, Paul H. 2, 28 Charles Gamier, Loretteville,
Quebec, Canada, G2A 2X8
l:Canadian citizenship
z:American citizenship
(ii) TITLE OF THE INVENTION: HIGHLY CONSERVED GENES AN THEIR USE
TO GENERATE SPECIES-SPECIFIC, GENUS-SPECIFIC AND UNIVERSAL
NUCLEIC ACID PROBES AND AMPLIFICATION PRIMERS TO RAPIDLY
DETECT AND IDENTIFY BACTERIAL, FUNGAL AND PARASITICAL
PATHOGENS FROM CLINICAL SPECIMENS FOR DIAGNOSIS
(iii) NUMBER OF SEQUENCES: 940
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE:
(B) STREET:
(C) CITY:
(D) STATE:
(E) COUNTRY:
(F) ZIP:
(v) COMPUTER READABLE:
(A) MEDIUM TYPE:
(B) COMPUTER:
(C) OPERATING:
(D) SOFTWARE:
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION:
(B) FILING DATE:
204
CA 02307010 2000-OS-19
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME:
(B) REGISTRATION NUMBER:
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE:
(B) TELEFAX:
205
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID N0: 1
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 750 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Acinetobacter baumannii
(B) STRAIN: ATCC 19606
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 1
CAAACTCGTG AGCACATCCT TCTTTCTCGT CAGGTAGGTG TACCTTACAT 50
CATCGTATTC TTAAACAAAT GCGACCTTGT TGATGACGAA GAATTACTTG 100
AATTAGTAGA AATGGAAGTA CGTGAACTTC TTTCTACTTA TGACTTCCCA 150
GGTGATGACA CTCCAGTAAT CCGTGGTTCA GCTCTTGCAG CGCTTAACGG 200
TGAAGCTGGT CCTTACGGTG AAGAATCAGT TCTTGCTCTT GTAGCAGCAC 250
TTGACTCTTA CATCCCAGAG CCAGAGCGTG CAATCGACAA AGCATTCTTG 300
ATGCCAATCG AAGACGTATT CTCAATTTCT GGTCGTGGTA CAGTAGTAAC 350
AGGCCGTGTT GAAGCTGGTA TCATCAAAGT TGGTGAAGAA GTAGAGATCG 400
TTGGTATTAA AGATACAGTT AAAACAACTG TAACTGGCGT AGAAATGTTC 450
CGTAAACTTC TTGACGAAGG CCGTGCAGGT GAGAACTGTG GTATCTTACT 500
TCGTGGTACT AAGCGTGAAG AAGTACAACG TGGTCAAGTA CTTGCTAAAC 550
CAGGTACAAT CAAGCCGCAC ACTAAATTCG ACGCAGAAGT ATACGTACTT 600
TCTAAAGAAG AAGGTGGTCG TCACACTCCA TTCTTAAATG GTTACCGTCC 650
ACAGTTCTAC TTCCGTACAA CTGACGTAAC TGGTGCRATC CAGTTGAAAG 700
AAGGCGTTGA AATGGTAATG CCAGGTGACA ACGTTGAAAT GTCAGTAGAA 750
2)INFORMATION FOR SEQ ID N0: 2
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Actinomyces meyeri
(B) STRAIN: ATCC 35568
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 2
CGGTGCGATC CTCGTGGTCG CCGCGACCGA CGGCCCCATG GCCCAGACCC 50
GCGAGCACGT CCTGCTCGCC CGTCAGGTCG GCGTTCCCAC CATCCTCATC 100
GCCCTCAACA AGTCCGACAT GGTTGACGAC GAGGAAATGA TGGAACTGGT 150
CGAGGAGGAG TGCCGCGACC TGCTGGAGTC CCAGGACTTC GATCGCGATG 200
CCCCGATCGT CCAGGTTTCC GCTCTGAAGG CCCTCGAGGG CGACGCGGAG 250
TGGGTTGCCA AGATCGAGGA GCTCATGGAG GCTGTGGATT CCTACATCCC 300
CACCCCCGAG CGCGATATGG ACAAGCCCTT CCTCATGCCG ATCGAGGACG 350
TCTTCACGAT CACAGGTCGT GGCACGGTCG TCACGGGGCG TGTTGAGCGT 400
GGCAAGCTGC CGATCAACTC CGAGGTCGAG ATCCTCGGTA TCCGTGATCC 450
206
CA 02307010 2000-OS-19
CCAGAAGACC ACGGTCACCG GCATCGAGAT GTTCCACAAG TCGATGGACG 500
AGGCATGGGC CGGCGAGAAC TGTGGCCTGC TGCTGCGCGG TACCAAGCGC 550
GATGAGGTTG AGCGCGGCCA GGTTGTGGCC ATTCCCGGCT CCATCACGCC 600
TCACACCGAG TTCGAGGGCC AGGTTTACAT CCTCAAGAAG GAAGAGGGCG 650
GCCGTCACAA CCCGTTCTTC TCGAACTACC GTCCGCAGTT CTACTTCCGT 700
ACCACGGACG TGACCGGCGT CATCACCCTC CCCGAGGGCA CCGACATGGT 750
CATGCCTGGC GACACCACCG AGATCTCCGT TCAGCTGATC CAGCCCATCG 800
CCATGGAGCC CGGCTGGGCT TCGCCA 826
2)INFORMATION FOR SEQ ID N0: 3
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Aerococcus viridans
(B) STRAIN: ATCC 11563
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 3
TGGTGCGATC TTAGTAGTAT CTGCTGCTGA TGGTCCAATG CCACAAACTC 50
GTGAGCACAT CCTTTTAGCT GGCCAAATCG GTGTTCCTGC ATTCGTAGTA 100
TTCTTAAACA AAGTTGACCA AGTTGACGAT GAAGAATTAC TAGAATTAGT 150
TGAAATGGAA GTTCGTGACT TATTATCTGA GTACAACTAC CCAGGTGACG 200
ATCTACCTGT AATCGCTGGT TCTGCTTTAT TAGCATTACA AGGCGATGAA 250
GCTCAAGAAG CTAAAATCAT GGAATTAATG GAAGCTGTAG ACTCTTACAT 300
TCCAGAACCA GAACGTGACA ACGACAAACC ATTCATGATG CCAATTGAGG 350
ATGTATTCTC AATCACTGGT CGTGGTACTG TTGCTACAGG TCGTGTTGAA 400
CGTGGTGAAG TTCGTACAGG TGACGAAGTT GACATCGTTG GTATTGCTGA 450
ACAAATCGGT AAATCAGTTG TAACTGGTGT TGAAATGTTC CGTAAAAACT 500
TAGACTACGC TCAAGCTGGT GACAACATCG GTGCATTATT ACGTGGTGTT 550
CAACGTGAAG ACATCCAACG TGGTCAAGTA TTGGCTGCTC CTGGTTCAAT 600
CACTCCACAT ACTAAATTTA AAGCGCAAGT TTACGTTTTA TCTAAAGAAG 650
AAGGTGGACG TCATACACCA TTCTTAACTA ACTACCGTCC ACAATTCTAC 700
TTCCGTACTA CTGACATTAC TGGTGTTATC ACTTTACCAG AAGACGTAGC 750
TATGGTTATG CCTGGTGACA ACGTTGATAT GGACGTTGAA TTGATTCACC 800
CAGTTGCGAT CGAAGATGGT ACTAAATTCT CTATC 835
2)INFORMATION FOR SEQ ID NO: 4
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 827 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Achromobacter xylosoxidans subsp.
denitrificans
207
CA 02307010 2000-OS-19
(B) STRAIN: ATCC 15173
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 4
CCTGGTGGTG TCGGCCGCTG ACGGCCCGAT GCCGCAAACG CGCGAACACA 50
TCCTGCTGAG CCGCCAGGTT GGCGTGCCGT ACATCATCGT CTTCCTGAAC 100
AAGGCCGACA TGGTTGACGA CGCCGAGCTG CTTGAGCTGG TGGAAATGGA 150
AGTTCGCGAR CTGCTGAGCA AGTACGACTT CCCGGGCGAC GACACCCCGA 200
TCGTGAAGGG TTCGGCCAAG CTGGCGCTGG AAGGCGACAA GGGCGAACTG 250
GGCGAACAGG CCATCATGGC GCTGGCCGCT GCGCTGGACT CGTACATCCC 300
GACGCCTGAG CGTGCCGTTG ACGGCGCGTT CCTGATGCCG GTTGAAGACG 350
TGTTCTCGAT CTCGGGTCGC GGCACCGTGG TGACCGGCCG TATCGAACGC 400
GGCATCATCA AGGTCGGCGA GGAAATCGAA ATCGTCGGTC TGGTGCCGAC 450
GGTGAAGACG ACCTGCACGG GCGTGGAAAT GTTCCGCAAG CTGCTGGACC 500
AAGGTCAAGC CGGCGACAAC GTGGGCATCC TRCTGCGCGG CACCAAGCGT 550
GAAGACGTCC AGCGCGGCCA GGTTCTGGCC AAGCCGGGCT CGATCACCCC 600
GCACACGGAC TTCACGTCCG AGGTGTACAT CCTGTCCAAG GAAGAAGGCG 650
GCCGTCACAC TCCGTTCTTC CAAGGCTATC GTCCCCAGTT CTACTTCCGC 700
ACGACGGACG TGACGGGCAC GATCGAGCTG CCGGCCGACA AGGA.AATGGT 750
CCTGCCGGGC GACAACGTGG CCATGACGGT CAAGCTGCTG GCTCCGATCG 800
CCATGGAAGA AGGCCTGCGT TCGCCAC 827
2)INFORMATION FOR SEQ ID N0: 5
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 823 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Anaerorhabdus furcosus
(B) STRAIN: ATCC 25662
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 5
TGGATCAATC CTAGTAGTTG CTGCAACTGA TGGACCAATG CCTCAA.ACTC 50
GTGAACATAT CTTACTTGCT CGTCAAGTAG GTGTTCCAAG AATGGTTGTA 100
TTCTTGAACA AATGCGACAT GGTTGAAGAT GAAGAATTAA TCGACCTTGT 150
TGAAATGGAA GTTCGTGAAC TTCTAAGTGC TTACGGTTTC GAAGGTGATG 200
ATACACCAGT TATCCGTGGT TCTGCATTAA AATCTCTTGA AGGAAATGCT 250
GATTGGGAAG CAAAAGTTGC TGAATTAATG GATGCAGTTG ACTCTTGGAT 300
TCCAACTCCA ACTCATGAAA CAGACAAACC ATTCTTAATG GCTGTTGAAG 350
ATGTATTCAC AATTACAGGT CGTGGTACAG TTGCTACTGG ACGTGTTGAA 400
CGTGGACACT TAAACCTTAA CGAAGAAGTT GAAATCGTTG GTATTCATGA 450
TACTAAGAAA TCAGTTGTTA CTGGTATCGA AATGTTCCGT AAATTATTAG 500
ACTATGCTGA AGCAGGAGAC AACATTGGTG CATTATTACG TGGTGTTTCT 550
CGTGATGAAA TCGAACGTGG ACAATGTCTA GCTAAACCTG GATCAGTTAC 600
TCCACATACA GCTTTCAAAG CTCAAGTATA CGTATTAACT AAAGAAGAAG 650
GTGGACGTCA TACACCATTC GTAACTAACT ACCGTCCTCA ATTCTATTTC 700
CGTACAACTG ACGTAACAGG AGTTGTTAAA CTTCCTGAAG GTACTGAAAT 750
GGTTATGCCT GGAGACAACA TCGAAATGAT CGTTGAATTA ATCGCTCCAA 800
TCGCTGTTGA ACAAGGAACT AAG 823
208
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID N0: 6
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 825 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Bacillus anthracis
(B) STRAIN: 4229
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 6
CGGCGGTATC TTAGTAGTAT CTGCTGCTGA TGGCCCAATG CCTCAAACTC 50
GTGAGCACAT CCTTCTTTCT CGTCAAGTAG GTGTACCTTA CATCGTTGTA 100
TTCTTAAACA AATGCGACAT GGTAGACGAC GAAGAATTAT TAGAATTAGT 150
AGAAATGGAA GTTCGCGACC TATTATCTGA ATACGGATTC CCAGGCGACG 200
ACATTCCTGT AATCAAAGGT TCTGCTCTTA AAGCTCTTCA AGGAGAAGCT 250
GATTGGGAAG CAAA.P~ATCATTGAATTAATG GCTGAAGTTG ATGCTTACAT 300
CCCAACTCCA GAACGTGAAA CTGACAAACC ATTCTTAATG CCTGTAGAGG 350
ACGTATTCTC TATCACAGGT CGTGGTACAG TTGCTACTGG TCGTGTTGAG 400
CGCGGTATCG TTAAAGTTGG TGACGTAGTA GAAATCATCG GTCTTGCTGA 450
AGAAAATGCT TCTACAACTG TAACTGGTGT AGAGATGTTC CGTAAACTTC 500
TTGACCAAGC TCAAGCTGGA GACAACATCG GTGCTTTACT TCGTGGGGTT 550
GCTCGTGAAG ACATCCAACG TGGACAAGTA CTTGCAAA.AAGCGGTTCTGT 600
AAAAGCTCAC GCTAAATTCA AAGCTGAAGT TTTCGTATTA TCTAAAGAAG 650
AAGGTGGACG TCACACTCCA TTCTTCGCTA ACTACCGTCC TCAGTTCTAC 700
TTCCGTACAA CTGACGTAAC TGGTATCATC CAATTACCAG AAGGTACTGA 750
AATGGTAATG CCTGGTGACA ACATCGAAAT GACTATCGAA CTTATCGCTC 800
CAATCGCTAT CGAAGAGGGA ACTAA 825
2)INFORMATION FOR SEQ ID N0: 7
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 829 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Bacillus cereus
(B) STRAIN: ATCC 14579
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 7
CGGCGGTATC TTAGTAGTAT CTGCTGCTGA TGGCCCAATG CCTCAAACAC 50
GTGAGCACAT CCTTCTTTCT CGTCAAGTAG GTGTTCCTTA CATCGTTGTA 100
TTCTTAAACA AATGCGACAT GGTAGATGAC GAAGAATTAT TAGAATTAGT 150
AGAAATGGAA GTTCGCGACC TATTATCTGA ATACGGATTC CCAGGCGACG 200
ACATTCCTGT AATCAAAGGT TCTGCTCTTA AAGCTCTTCA AGGAGAAGCT 250
GATTGGGAAG CAAAAATCAT TGAATTAATG GCTGAAGTTG ATGCTTACAT 300
CCCAACTCCA GAACGTGAAA CTGACAAACC ATTCTTAATG CCTGTAGAGG 350
ACGTATTCTC TATCACAGGT CGTGGTACAG TTGCTACTGG TCGTGTTGAG 400
209
CA 02307010 2000-OS-19
CGCGGTATCG TTAAAGTTGG TGACGTAGTA GAAATCATCG GTCTTGCTGA 450
AGAAAATGCT TCTACAACTG TAACTGGTGT AGAGATGTTC CGTAAACTTC 500
TTGACCAAGC TCAAGCTGGA GACAACATCG GTGCTTTACT TCGTGGGGTT 550
GCTCGTGAAG ACATCCAACG TGGACAAGTA CTTGCAAAAA GCGGTTCTGT 600
AAAAGCTCAC GCTAAATTCA AAGCTGAAGT TTTCGTATTA TCTAAAGAAG 650
AAGGTGGACG TCACACTCCA TTCTTCGCTA ACTACCGTCC TCAGTTCTAC 700
TTCCGTACAA CTGACGTAAC TGGTATCATC CAATTACCAG AAGGTACTGA 750
AATGGTAATG CCTGGTGACA ACATTGAAAT GACTATCGAA CTTATCGCTC 800
CAATCGCTAT CGAAGAGGGA ACTAAATTC 829
2)INFORMATION FOR SEQ ID NO: 8
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 818 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Bacteroides distasonis
(B) STRAIN: ATCC 8503
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 8
CGGTGCTATC ATCGTAGTTG CTGCTACTGA TGGTCCTATG CCTCAAACTC 50
GCGAGCACAT CCTTTTGGCT CGTCAGGTAA ACGTTCCGAG ATTGGTTGTA 100
TTCATGAACA AGTGTGACAT GGTTGACGAC GAGGAAATGT TGGAATTGGT 150
TGAGATGGAG ATGAGAGAGT TGCTTTCATT CTATCAATTC GACGGTGACA 200
ACACTCCGAT CATCCGTGGT TCTGCTCTTG GTGCATTGAA CGGTGATGCT 250
CAATGGGAAG ATAAAGTAAT GGAGTTGATG GAAGCTTGTG ATACTTGGAT 300
TCCTCTGCCT CCGCGCGAAA TCGACAAGCC GTTCTTGATG CCGGTTGAGG 350
ACGTATTCTC AATCACGGGT CGTGGTACTG TTGCTACAGG TCGTATCGAG 400
ACAGGTATTG TTAAGGTTGG TGAGGAAGTT CAGATCATCG GTCTTGGCGC 450
TGCTGGTAAG AAATCTGTTG TTACAGGTGT TGAGATGTTC CGTAAGTTAT 500
TGGATCAAGG TGAGGCTGGT GATAACGTTG GTTTGTTGCT TCGCGGTATC 550
GATAAGAATG AGATCAAGCG TGGTATGGTA ATCTGCCACC CGGGTCAGGT 600
TAAAGAGCAT TCTAAGTTCA AGGCTGAGGT TTATATCTTG AAGAAAGAGG 650
AAGGTGGTCG TCACACTCCG TTCCACAACA AATATCGTCC TCAGTTCTAT 700
ATCCGTACAT TGGATGTAAC TGGTGAGATC ACTTTGCCGG AAGGAACTGA 750
AATGGTAATG CCGGGTGATA ACGTAACGAT CGAGGTTGAG TTGATCTATC 800
CGGTAGCATG TAGCGTAG 818
2)INFORMATION FOR SEQ ID NO: 9
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 639 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus casseliflavus
210
CA 02307010 2000-OS-19
(B) STRAIN: 8763
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 9
GGTCCTATGC CTCAAACACG TGAACACATC TTGTTATCAC GTAACGTTGG 50
TGTACCATAC ATCGTTGTTT TCTTAAACAA AATGGATATG GTTGATGACG 100
AAGAATTACT AGAATTAGTT GAAATGGAAG TTCGTGACTT ATTGTCAGAA 150
TATGACTTCC CAGGCGACGA TGTTCCTGTA ATCGCTGGTT CTGCTTTGAA 200
AGCTCTTGAA GGCGATGCTT CATACGAAGA AAAAATCATG GAATTAATGG 250
CTGCAGTTGA CGAATACGTT CCAACTCCAG AACGTGACAC TGACAAACCA 300
TTCATGATGC CAGTCGAAGA CGTATTCTCA ATCACTGGAC GTGGTACTGT 350
TGCTACAGGC CGTGTTGAAC GTGGACAAGT TCGCGTTGGT GACGAAGTTG 400
AAATCGTTGG TATTGCTGAA GAAACTGCTA AAACAACTGT AACTGGTGTT 450
GAAATGTTCC GTAAATTGTT AGACTATGCT GAAGCAGGGG ATAACATTGG 500
TGCATTGCTA CGTGGTGTTG CTCGTGAAGA CATCCAACGT GGACAAGTAT 550
TGGCTAAAGC TGGTACAATC ACACCTCATA CAAAATTTAA AGCTGAAGTT 600
TACGTTTTAA CAAAAGAAGA AGGTGGACGT CACACACCA 639
2)INFORMATION FOR SEQ ID N0: 10
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 692 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Staphylococcus saprophyticus
(B) STRAIN: CSG 197
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 10
GAACACATTC TTTTATCACG TAACGTTGGT GTTCCAGCAT TAGTTGTATT 50
CTTAAACAAA GTTGACATGG TTGACGATGA AGAATTATTA GAATTAGTAG 100
AAATGGAAGT TCGTGACTTA TTAAGCGAAT ATGACTTCCC AGGTGACGAT 150
GTACCTGTAA TCTCTGGTTC TGCATTAAAA GCTTTAGAAG GCGACGCTGA 200
CTATGAGCAA AAAATCTTAG ACTTAATGCA AGCTGTTGAT GACTTCATTC 250
CAACACCAGA ACGTGATTCT GACAAACCAT TCATGATGCC AGTTGAGGAC 300
GTATTCTCAA TCACTGGTCG TGGTACTGTT GCTACAGGCC GTGTTGAACG 350
TGGTCAAATC AAAGTCGGTG AAGAAATCGA AATCATCGGT ATGCAAGAAG 400
AATCAAGCAA AACAACTGTT ACTGGTGTAG AAATGTTCCG TAAATTATTA 450
GACTACGCTG AAGCTGGTGA CAACATTGGT GCATTATTAC GTGGTGTTTC 500
ACGTGATGAC GTACAACGTG GTCAAGTTTT AGCTGCTCCT GGTACTATTA 550
CACCACATAC AAAATTCAAA GCGGATGTTT ACGTTTTATC TAAAGATGAA 600
GGTGGTCGTC ATACACCATT CTTCACTAAC TACCGCCCAC AATTCTATTT 650
CCGTACTACT GACGTAACTG GTGTTGTTAA CTTACCAGAA GG 692
2)INFORMATION FOR SEQ ID NO: 11
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 821 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
211
CA 02307010 2000-OS-19
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Bacteroides ovatus
(B) STRAIN: ATCC 8483
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 11
CGGTGCTATC ATCGTTTGTG CTGCAACTGA TGGTCCGATG CCTCAAACTC 50
GCGAACACAT TCTGTTAGCT CGTCAGGTAA ACGTACCTCG TCTGGTTGTA 100
TTCTTGAACA AATGCGATAT GGTAGACGAC GAAGAAATGT TGGAACTCGT 150
TGAAATGGAA ATGAGAGAAC TCCTTTCATT CTATGATTTC GATGGTGACA 200
ATACTCCTAT CATCCGTGGT TCTGCTCTTG GCGCATTGAA CGGTGTTGAA 250
AAATGGGAAG ACAAAGTTAT GGAACTGATG GATGCAGTTG ATAACTGGAT 300
TCCACTGCCT CCGCGCGATG TTGATAAACC ATTCTTGATG CCGGTTGAAG 350
ACGTGTTCTC TATCACAGGT CGTGGTACTG TAGCAACAGG TCGTATCGAA 400
ACAGGTGTCA TCCACGTTGG TGATGAAGTC GAAATTCTTG GTTTAGGTGA 450
AGATAAGAAA TCAGTTGTAA CTGGTGTTGA AATGTTCCGT AAACTGTTGG 500
ATCAAGGTGA AGCTGGTGAC AACGTAGGTC TTTTGCTTCG TGGTATTGAC 550
AAGAACGAAA TCAAACGTGG TATGGTTCTT TGTAAACCAG GTCAGATTAA 600
ACCGCACTCT AAATTCAAAG CTGAGGTTTA TATCTTGAAG AAAGAAGAAG 650
GTGGTCGTCA CACTCCGTTC CACAACAAAT ACCGTCCTCA GTTCTACTTG 700
CGTACTATGG ACTGTACAGG TGAAATCACT TTGCCGGAAG GAACAGAAAT 750
GGTAATGCCG GGTGATAACG TAACTATTAC AGTTGAGTTG ATTTACCCAG 800
TAGCATTGAA CCCGGGCTTC G 821
2)INFORMATION FOR SEQ ID N0: 12
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 838 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Bartonella henselae
(B) STRAIN: ATCC 49882
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 12
TGGTGCGATT TTGGTTGTTT CAGCTGCTGA TGGTCCGATG CCTCAAACAC 50
GTGAGCATAT TCTTCTTGCC CGTCAGGTTG GTGTTCCAGC GATTGTTGTT 100
TTTCTTAATA AGGTTGATCA GGTTGATGAT GCTGAGCTTT TGGAGCTTGT 150
TGAGCTTGAA GTTCGGGAGT TATTGTCGAA ATATGATTTT CCAGGAGACG 200
ATATTCCGAT CGTTAAAGGT TCTGCTTTGG CAGCGCTTGA AGATAAAGAT 250
AAAAGCATTG GTGAAGATGC GGTTCGTCTT TTGATGAGTG AAGTTGATAA 300
TTATATACCG ACGCCTGAAC GTCCTGTTGA TCAGCCGTTT TTGATGCCAA 350
TTGAAGATGT TTTTTCGATT TCGGGTCGTG GAACTGTTGT GACGGGTCGT 400
GTTGAGCGTG GTGTTATTAA GGTTGGTGAA GAAGTTGAGA TTATCGGCAT 450
TCGTCCAACT TCTAAGACAA CAGTTACAGG GGTTGAAATG TTCCGCAAGC 500
TTTTAGATCA GGGGCAAGCG GGTGATAATA TTGGAGCGCT GCTTCGTGGT 550
ATTGATCGTG AAGGGATTGA GCGTGGACAA GTTTTGGCGA AGCCTGCTTC 600
GGTTACACCT CATACGAGAT TTAAAGCAGA GGCTTACATT TTGACGAAAG 650
ATGAAGGTGG TCGTCATACT CCATTTTTCA CGAATTATCG TCCTCAGTTT 700
TATTTCCGTA CTACGGATGT AACGGGAATT GTTACGCTTC CAGAAGGTAC 750
212
CA 02307010 2000-OS-19
AGAGATGGTT ATGCCTGGTG ATAATGTTGC TATGGATGTC TCTCTGATTG 800
TTCCAATTGC CATGGAAGAA AAACTTCGTT TTGCTATC 838
2)INFORMATION FOR SEQ ID N0: 13
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 839 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Bifidobacterium adolescentis
(B) STRAIN: ATCC 15703
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 13
TGGCGCCATC CTTGTTGTGG CCGCCACCGA CGGCCCGATG GCTCAGACCC 50
GCGAGCACGT GCTGCTCGCT CGTCAGGTGG GCGTCCCGAA GATCCTCGTC 100
GCTCTGAACA AGTGCGATAT GGTCGACGAC GACGAGCTCA TCGAGCTCGT 150
TGAGGAAGAG GTCCGTGACC TCCTCGACGA AAATGGCTTC GATCGCGATT 200
GCCCGGTCAT CCACGTGTCC GCTTACGGCG CACTGCACGA TGACGCTCCG 250
GACCACGAGA AGTGGGTTGA GCAGATCAAG AAGCTCATGG ACGCCGTCGA 300
TGACTACATC CCGACCCCGG TCCACGATCT GGACAAGCCG TTCCTGATGC 350
CGATCGAAGA TGTCTTCACC ATCTCCGGCC GTGGCACCGT GGTGACCGGC 400
CGTGTCGAGC GTGGTAAGCT CCCGGTCAAC TCCAACGTCG AGATCGTCGG 450
CATCCGTCCG ACCCAGACCA CCACCGTCAC CTCCATCGAG ACCTTCCACA 500
AGCAGATGGA CGAGTGCGAG GCTGGCGACA ACACCGGTCT GCTGCTCCGC 550
GGCATCAACC GTGACCAGGT CGAGCGTGGC CAGGTTCTGG CTGCTCCGGG 600
CTCCGTGACC CCGCACACCA AGTTCGAGGG CGAAGTCTAC GTGCTGACCA 650
AGGACGAAGG CGGCCGTCAC TCGCCGTTCT TCTCCAACTA CCGTCCGCAG 700
TTCTACTTCC GTACCACCGA CGTCACCGGC GTCATCACCC TGCCGGAAGG 750
CGTTGAGATG GTGCAGCCGG GCGATCACGC TACCTTCGGC GTTGAGCTGA 800
TCCAGCCGAT CGCTATGGAA GAGGGCCTGA CCTTCGCAG 839
2)INFORMATION FOR SEQ ID NO: 14
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 839 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Bifidobacterium dentium
(B) STRAIN: ATCC 27534
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 14
TGGCGCTATC CTCGTTGTGG CCGCCACCGA CGGCCCGATG GCTCAGACCC 50
GCGAGCACGT GCTGCTCGCT CGTCAGGTGG GCGTGCCGCG TATCCTCGTC 100
GCCCTGAACA AGTGCGATAT GGTCGACGAC GAAGAGCTCA TCGAGCTCGT 150
213
CA 02307010 2000-OS-19
TGAGGAAGAG GTCCGTGACC TCCTCGACGA AAACGGCTTC GATCGCGATT 200
GCCCGGTCAT CCACACCTCC GCCTACGGCG CGCTGCACGA TGACGCTCCG 250
GACCACGACA AGTGGGTTGA GTCCGTCAAG GAACTCATGA AGGCCGTCGA 300
CGAGTACATC CCGACCCCGA CCCACGATCT GGACAAGCCG TTCCTGATGC 350
CGATCGAAGA TGTGTTCACC ATCTCCGGCC GTGGCACCGT GGTTACCGGC 400
CGTGTCGAGC GTGGTAAGCT CCCGGTCAAC TCCAACGTTG AGATCGTCGG 450
CATCCGTCCG ACCCAGACCA CCACCGTCAC CTCCATCGAG ACCTTCCACA 500
AGCAGATGGA CGAGTGCGAG GCTGGCGACA ACACCGGTCT GCTGCTCCGC 550
GGCATCAACC GTGACCAGGT CGAGCGTGGC CAGGTTCTGG CTGCTCCGGG 600
CTCCGTGACC CCGCACACCA AGTTCGAGGG CGAAGTCTAC GTGCTGACCA 650
AGGACGAAGG CGGCCGTCAC TCGCCGTTCT TCTCCAACTA CCGTCCGCAG 700
TTCTACTTCC GTACCACCGA CGTCACCGGC GTCATCACCC TGCCGGAAGG 750
CGTTGAGATG GTGCAGCCGG GCGATCACGC TACCTTCGGC GTTGAGCTGA 800
TCCAGCCGAT CGCTATGGAA GAGGGCCTGA CCTTCGCAG 839
2)INFORMATION FOR SEQ ID NO: 15
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 838 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Brucella abortus
(B) STRAIN: 52308
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 15
TGGCGCGATC CTGGTGGTTT CGGCTGCTGA CGGCCCGATG CCGCAGACCC 50
GCGAGCACAT CCTGCTTGCC CGTCAGGTTG GCGTTCCGGC GATCGTCGTG 100
TTCCTCAACA AGTGCGACCA GGTTGACGAT GCAGAACTGC TCGAACTGGT 150
TGAACTGGAA GTGCGCGAAC TTCTGTCGAA GTACGAATTC CCCGGCGACG 200
AAATCCCGAT CATCAAGGGC TCGGCTCTTG CTGCTCTGGA AGATTCTTCC 250
AAGGAACTGG GCGAAGATGC CATCCGCAAC CTGATGGACG CGGTTGACAG 300
CTACATTCCG ACCCCGGAAC GCCCGATCGA CCAGCCGTTC CTGATGCCGA 350
TCGAAGACGT GTTCTCGATC TCCGGCCGTG GTACGGTTGT GACGGGTCGC 400
GTTGAGCGCG GTATCGTTAA GGTCGGTGAA GAAGTTGAAA TCGTCGGCAT 450
CAAGGCGACG ACGAAGACCA CGGTTACCGG CGTTGAAATG TTCCGCAAGC 500
TGCTCGACCA GGGCCAGGCT GGCGACAACA TTGGCGCGCT GATCCGCGGC 550
GTTGGCCGTG AAGACGTTGA ACGCGGCCAG GTTCTCTGCA AGCCGGGTTC 600
TGTGAAGCCG CACACCAAGT TTAAGGCAGA AGCCTATATT CTGACCAAGG 650
ACGAAGGTGG CCGTCATACG CCGTTCTTCA CCAACTACCG TCCGCAGTTC 700
TACTTCCGTA CGACGGACGT GACGGGTGTT GTGACGCTTC CGGCTGGCAC 750
GGAAATGGTC ATGCCTGGCG ATAACGTCGC CATGGACGTT ACCCTGATCG 800
TGCCGATCGC CATGGAAGAG AAGCTTCGCT TCGCTATC 838
2)INFORMATION FOR SEQ ID NO: 16
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 771 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
214
CA 02307010 2000-OS-19
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Burkholderia cepacia
(B) STRAIN: LSPQ 2217
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 16
GGCAGCAGAC GGCCCGATGC CGCAAACGCG TGAGCACATC CTGCTGGCGC 50
GTCAGGTTGG CGTTCCGTAC ATCATCGTGT TCCTGAACAA GTGCGACATG 100
GTGGACGACG CCGAACTGCT CGAGCTGGTC GAGATGGAAG TTCGCGAACT 150
CCTGTCGAAG TACGACTTCC CGGGCGACGA CACGCCGATC GTGAAGGGTT 200
CGGCGAAGCT GGCGCTGGAA GGCGACACGG GCGAGCTGGG CGAAGTGGCG 250
ATCATGAGCC TGGCCGACGC GCTGGACACG TACATCCCGA CGCCGGAGCG 300
TGCAGTTGAC GGCGCGTTCC TGATGCCGGT GGAAGACGTG TTCTCGATCT 350
CGGGCCGCGG TACGGTGGTG ACGGGTCGTG TCGAGCGCGG CATCGTGAAG 400
GTCGGCGAAG AAATCGAAAT CGTCGGTATC AAGCCGACGG TGAAGACGAC 450
CTGCACGGGC GTTGAAATGT TCCGCAAGCT GCTGGACCAA GGTCAAGCAG 500
GCGACAACGT TGGTATCCTG CTGCGCGGCA CGAAGCGTGA AGACGTGGAG 550
CGTGGCCAGG TTCTGGCGAA GCCGGGTTCG ATCACGCCGC ACACGCACTT 600
CACGGCTGAA GTGTACGTGC TGAGCAAGGA CGAAGGCGGC CGTCACACGC 650
CGTTCTTCAA CAACTACCGT CCGCAGTTCT ACTTCCGTAC GACGGACGTG 700
ACGGGCTCGA TCGAGCTGCC GAAGGACAAG GAAATGGTGA TGCCGGGCGA 750
CAACGTGTCG ATCACGGTGA A 771
2)INFORMATION FOR SEQ ID N0: 17
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 829 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Cedecea davisae
(B) STRAIN: ATCC 33431
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 17
GGCGCTATCC TGGTTGTTGC TGCGACTGAT GGCCCAATGC CACAGACCCG 50
TGAGCACATC CTGCTGGGTC GTCAGGTTGG CGTTCCGTAC ATCATCGTGT 100
TCCTGAACAA ATGCGACATG GTTGATGACG AAGAGCTGCT GGAACTGGTA 150
GAAATGGAAG TTCGTGAACT TCTGTCCCAG TACGACTTCC CGGGCGACGA 200
TACTCCAATC GTTCGTGGTT CTGCTCTGAA AGCGCTGGAA GGCGAAGCAG 250
AGTGGGAAGC TAAAATCGTT GAGCTGGCTG GCTACCTGGA TTCTTACATC 300
CCTGAGCCAG AGCGTGCTAT CGATAAGCCG TTCCTGCTGC CAATCGAAGA 350
CGTATTCTCC ATCTCCGGCC GTGGTACCGT TGTTACCGGT CGTGTAGAGC 400
GCGGTATCAT CAAAGTTGGT GAAGAAGTTG AAATCGTTGG TATCAAAGAT 450
ACTGCGAAAT CTACCTGTAC CGGCGTTGAA ATGTTCCGCA AACTGCTGGA 500
CGAAGGCCGT GCTGGTGAGA ACGTTGGTGT TCTGCTGCGT GGTATCAAAC 550
GTGAAGAAAT CGAACGTGGT CAGGTACTGG CTAAGCCAGG CTCTATCAAG 600
CCACACACCA AGTTCGAATC TGAAGTGTAC ATCCTGTCCA AAGACGAAGG 650
CGGCCGTCAT ACTCCGTTCT TCAAAGGCTA CCGTCCACAG TTCTACTTCC 700
GTACAACTGA CGTGACCGGC ACCATCGAAC TGCCAGAAGG CGTTGAGATG 750
GTAATGCCTG GCGACAACAT CAAAATGGTT GTTACCCTGA TCCACCCAAT 800
215
CA 02307010 2000-OS-19
CGCGATGGAT GACGGTCTGC GTTTCGCAA 829
2)INFORMATION FOR SEQ ID NO: 18
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 824 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Cedecea neteri
(B) STRAIN: ATCC 33855
(xi)SEQUENCE
DESCRIPTION:
SEQ ID NO:
18
CGCTATCCTG GTTGTTGCTG CGACTGACGG CCCTATGCCT CAGACCCGTG 50
AGCACATCCT GCTGGGTCGT CAGGTTGGCG TTCCTTACAT CATCGTGTTC 100
CTGAACAAAT GTGACATGGT TGATGACGAA GAGCTGCTGG AGCTGGTTGA 150
AATGGAAGTT CGTGAACTTC TGTCTCAGTA CGACTTCCCG GGCGATGACA 200
CTCCAATCAT CCGTGGTTCT GCTCTGAAAG CGCTGGAAGG CGAAGCAGAG 250
TGGGAAGCTA AAATYGTTGA GCTGGCTGGC TTCCTGGATT CCTACATCCC 300
AGAACCAGTA CGTGCAATCG AYCTGCCGTT CCTGCTGCCA ATCGAAGACG 350
TATTCTCCAT CTCCGGCCGT GGTACCGTTG TTACCGGTCG TGTAGAGCGC 400
GGTATCGTTA AAGTGGGCGA AGAAGTAGAA ATCGTTGGTA TCAAAGATAC 450
TGCGAAATCT ACCTGTACCG GCGTTGAAAT GTTCCGCAAA CTGCTGGACG 500
AAGGCCGTGC TGGTGAGAAC GTTGGTGTTC TGCTGCGTGG TATCAAACGT 550
GAAGAAATCG AACGTGGTCA GGTTCTGGCT AAGCCAGGCT CTATCAAGCC 600
GCACACCAAG TTCGAATCTG AAGTGTACAT CCTGTCCAAA GACGAAGGCG 650
GCCGTCATAC TCCGTTCTTC AAAGGCTACC GTCCACAGTT CTACTTCCGT 700
ACAACTGACG TGACCGGTAC CATCGAACTG CCAGAAGGCG TAGAGATGGT 750
AATGCCAGGC GACAACATCA AAATGGTTGT TACCCTGATC CACCCAATCG 800
CGATGGACGA CGGTCTGCGT TTCG 824
2)INFORMATION FOR SEQ ID NO: 19
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 827 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Cedecea lapagei
(B) STRAIN: ATCC 33432
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 19
CGCTATTCTG GTTGTTGCTG CAACTGACGG CCCTATGCCT CAGACCCGTG 50
AGCACATCCT GCTGGGTCGC CAGGTTGGCG TTCCTTACAT CATCGTGTTC 100
CTGAACAAAT GTGACATGGT TGATGACGAA GAGCTGCTGG AGCTGGTAGA 150
AATGGAAGTT CGTGAACTTC TGTCTCAGTA CGACTTCCCA GGCGATGATA 200
216
CA 02307010 2000-OS-19
CCCCAATCAT CCGTGGTTCT GCTCTGAAAG CGCTGGAAGG CGAAGCAGAG 250
TGGGAAGCTA AAATCGTTGA GCTGGCTGGC TTCCTGGATT CCTACATCCC 300
AGAACCAGTA CGTGCAATCG ACCTGCCGTT CCTGCTGCCA ATCGAAGACG 350
TATTCTCCAT CTCCGGCCGT GGTACCGTTG TKACCGGTCG TGTAGAGCGC 400
GGTATCGTTA AAGTGGGCGA AGAAGTAGAA ATCGTTGGTA TCAAAGATAC 450
TGCGAAATCT ACCTGTACTG GCGTTGAAAT GTTCCGCAAA CTGCTGGACG 500
AAGGCCGTGC TGGTGAGAAC GTTGGTGTTC TGCTGCGTGG TATCAAACGT 550
GAAGAAATCG AACGTGGTCA GGTTCTGGCT AAGCCAGGCT CTATCAAGCC 600
GCACACCAAG TTCGAATCTG AAGTGTACAT CCTGTCCAAA GACGAAGGCG 650
GCCGTCATAC TCCGTTCTTC AARGGCTACC GTCCACAGTT CTACTTCCGT 700
ACCACTGACG TGACCGGTAC CATCGAACTG CCAGAAGGCG TAGAGATGGT 750
AATGCCAGGT GACAACATCA AAATGGTTGT TACCCTGATC CACCCAATCG 800
CGATGGACGA CGGTCTGCGT TTCGCAA 827
2)INFORMATION FOR SEQ ID NO: 20
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 831 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Chlamydia pneumoniae
(B) STRAIN: CWL 029
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 20
GCGGAGCTAT CCTAGTCGTT TCAGCTACAG ACGGAGCTAT GCCACAAACT 50
AAAGAACATA TCTTGCTAGC TCGCCAGGTT GGAGTTCCTT ATATCGTTGT 100
TTTCTTGAAT AAAGTAGATA TGATCTCTCA AGAAGATGCT GAACTTATTG 150
ACCTTGTTGA GATGGAACTT AGTGAGCTTC TTGAAGAAA.AAGGCTACAAA 200
GGATGCCCTA TTATCCGTGG TTCTGCTTTG AAAGCTCTTG AAGGTGATGC 250
AAATTATATC GAAAAAGTTC GAGAACTTAT GCAAGCTGTG GATGACAACA 300
TCCCTACACC AGAAAGAGAA ATTGATAAGC CTTTCTTAAT GCCTATCGAA 350
GACGTATTCT CAATCTCTGG TCGTGGTACT GTGGTTACAG GAAGAATCGA 400
GCGTGGAATC GTTAAAGTTT CTGATAAAGT TCAGCTCGTG GGATTAGGAG 450
AGACTAAAGA AACAATCGTT ACTGGAGTCG AAATGTTCAG GAAAGAACTT 500
CCTGAAGGTC GTGCAGGAGA AAACGTTGGT TTACTCCTCA GAGGTATTGG 550
AAAGAACGAT GTTGAAAGAG GTATGGTGGT TTGTCAGCCT AACAGCGTGA 600
AGCCTCATAC GAAATTTAAG TCAGCTGTTT ACGTTCTTCA GAAAGAAGAA 650
GGCGGACGTC ATAAGCCTTT CTTCAGCGGA TACAGACCTC AGTTCTTCTT 700
CCGTACTACA GACGTGACAG GAGTCGTAAC TCTTCCTGAA GGAACTGAAA 750
TGGTAATGCC TGGAGATAAC GTTGAGCTTG ATGTTGAGCT CATTGGAACA 800
GTTGCTCTTG AAGAAGGAAT GAGATTTGCA A 831
2)INFORMATION FOR SEQ ID NO: 21
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
217
CA 02307010 2000-OS-19
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Chlamydia psittaci
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 21
TGGAGCGATT CTCGTTGTTT CCGCTACTGA CGGTGCGATG CCTCAGACCA 50
AAGAACATAT TCTTTTGGCG AGACAGGTTG GTGTTCCTTA CATCGTTGTT 100
TTCCTTAACA AAATCGATAT GATTTCTCAA GAAGATGCTG AGCTCGTAGA 150
CTTAGTTGAA ATGGAATTGT CCGAACTTCT AGAAGAAAAA GGTTATAAAG 200
GTTGCCCAAT TATCCGTGGT TCTGCTTTGA AAGCCTTAGA AGGTGATGCA 250
AGCTACGTTG AAAAAATTCG CGAGTTAATG CAAGCAGTGG ATGATAACAT 300
CCCTACTCCA GAGCGTGAAG TTGATAAGCC TTTCTTAATG CCTATCGAAG 350
ACGTATTCTC TATTTCTGGT CGTGGTACTG TGGTCACAGG ACGTATCGAG 400
CGTGGAATCG TTAAAGTGGG TGATAAAGTA CAGATTGTTG GTTTAAGAGA 450
TACTAGAGAG ACAATTGTTA CCGGTGTGGA AATGTTCAGA AAAGAACTTC 500
CAGAAGGTCA AGCAGGGGAA AACGTTGGTT TGCTCCTCAG AGGTATCGGT 550
AAGAATGACG TTGAACGTGG TATGGTTATC TGCCAACCTA ATAGCGTGAA 600
ATCTCACACA CAATTTAAAG GTGCTGTCTA CATTCTACAA AAAGAAGAGG 650
GTGGACGTCA TAAACCTTTC TTTACCGGAT ACAGACCTCA GTTCTTCTTC 700
CGTACAACAG ATGTTACAGG TGTTGTAACT CTCCCAGAAG GTACAGAGAT 750
GGTTATGCCA GGCGATAACG TTGAATTCGA AGTTCAATTA ATTAGCCCAG 800
TAGCTCTAGA AGAAGGTATG AGATTT 826
2)INFORMATION FOR SEQ ID N0: 22
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 822 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Chlamydia trachomatis
(B) STRAIN: LGV 12
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 22
GGGGCTATTC TAGTAGTTTC TGCAACAGAC GGAGCTATGC CTCAAACTAA 50
AGAGCATATT CTTTTGGCAA GACAAGTTGG GGTTCCTTAC ATCGTTGTTT 100
TTCTCAATAA AATTGACATG ATTTCCGAAG AAGACGCTGA ATTGGTCGAC 150
TTGGTTGAGA TGGAGTTGGC TGAGCTTCTT GAAGAGAAAG GATACAAAGG 200
GTGTCCAATC ATCAGAGGTT CTGCTCTGAA AGCTTTGGAA GGGGATGCTG 250
CATACATAGA GAA.AGTTCGAGAGCTAATGC AAGCCGTCGA TGATAATATC 300
CCTACTCCAG AAAGAGAAAT TGACAAGCCT TTCTTAATGC CCATTGAGGA 350
CGTGTTCTCT ATCTCCGGAC GAGGAACTGT AGTAACTGGA CGTATTGAGC 400
GTGGAATTGT TAAAGTTTCC GATAAAGTTC AGTTGGTCGG TCTTAGAGAT 450
ACTAAAGAAA CGATTGTTAC TGGGGTTGAA ATGTTCAGAA AAGAACTCCC 500
AGAAGGTCGT GCAGGAGAGA ATGTTGGATT GCTCCTCAGA GGTATTGGTA 550
AGAACGATGT GGAAAGAGGA ATGGTTGTTT GCTTGCCAAA CAGTGTTAAA 600
CCTCATACAC GGTTTAAGTG TGCTGTTTAC GTTCTGCAAA AAGAAGAAGG 650
TGGACGACAT AAGCCTTTCT TCACAGGATA TAGACCTCAA TTCTTCTTCC 700
GTACAACAGA CGTTACAGGT GTGGTAACTC TGCCTGAGGG AGTTGAGATG 750
GTCATGCCTG GGGATAACGT TGAGTTTGAA GTGCAGTTGA TTAGCCCTGT 800
GGCTTTAGAA GAAGGTATGA GA 822
218
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID NO: 23
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Chryseobacterium meningosepticum
(B) STRAIN: CDC B7681
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 23
CGGAGCTATC TTAGTATGTG CTGCTACAGA TGGTCCAATGCCTCAAACTA 50
GAGAACACAT CCTACTTTGC CGTCAGGTAA ACGTACCTAGAATTGTTGTG 100
TTCATGAACA AAGTTGACAT GGTAGATGAT CCAGAATTGTTAGAGCTTGT 150
TGAGCTTGAA CTTAGAGATC TATTATCTAC TTACGAATATGATGGTGATA 200
ACTCTCCAGT AATTCAAGGT TCTGCTCTTG GTGCTCTTAACGGTGATGCT 250
AAGTGGGTAG CTACTGTAGA AGCTCTAATG GATGCTGTTGATACTTGGAT 300
CGAGCAACCA GTAAGAGATT CTGATAAGCC ATTCCTTATGCCAATCGAAG 350
ACGTATTCTC TATTACAGGT AGAGGTACTG TAGCAACTGGTAGAATCGAG 400
GCTGGTGTAA TCAACACAGG TGATCCTGTT GACATCGTAGGTATGGGTGA 450
CGAGAAGTTA ACTTCTACTA TTACAGGTGT TGAGATGTTTAGAAAAATCC 500
TAGACAGAGG TGAAGCTGGT GATAACGTAG GTCTATTGTTGAGAGGTATT 550
GAAAAGACTG ACATCAAGAG AGGTATGGTT ATCGCTAAGAAAGATTCAGT 600
TAAGCCACAC AAGAAATTCA AAGCTGAGGT TTATATCCTTTCTAAAGAAG 650
AAGGTGGACG TCACACTCCA TTCCACAACA AATACCGTCCTCAGTTCTAT 700
GTAAGAACTA CTGACGTTAC AGGTGAAATC TTCTTACCAGAAGGTGTAGA 750
AATGGTAATG CCTGGTGATA ACTTAACTAT CACTGTAGAATTGTTACAAC 800
CAATCGCTCT TAACGAGGGT CTTAGATTCG CGATC 835
2)INFORMATION FOR SEQ ID N0: 24
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 816 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Citrobacter amalonaticus
(B) STRAIN: ATCC 25405
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 24
CGGCGCGATC CTGGTTGTTG CTGCGACTGA CGGCCCGATG CCGCAGACTC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCGTA CATCATCGTG 100
TTCCTGAACA AATGCGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
AGAAATGGAA GTTCGTGAAC TTCTGTCTCA GTACGATTTC CCGGGCGACG 200
ACACCCCGAT CGTTCGTGGT TCTGCTCTGA AAGCGCTGGA AGGCGACGCA 250
219
CA 02307010 2000-OS-19
GAGTGGGAAG CGAAAATCAT CGAACTGGCC GGCTTCCTGG ATTCTTACAT 300
CCCGGAACCA GAGCGTGCGA TTGACAAGCC GTTCCTGCTG CCGATCGAAG 350
ACGTATTCTC CATCTCCGGT CGTGGTACCG TTGTTACCGG TCGTGTAGAA 400
CGCGGTATCA TCAAAGTGGG CGAAGAAGTT GAAATCGTTG GTATCAAAGA 450
GACTGCCAAG TCTACCTGTA CTGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGCCG TGCGGGTGAG AACGTTGGTG TTCTGCTGCG TGGTATCAAA 550
CGTGAAGAAA TCGAACGTGG TCAGGTACTG GCTAAGCCGG GCWCCATCAA 600
GCCGCACACC ATGTTCGAAT CYGAAGTGTA CATCCTGTCC AAAGACGAAG 650
GCGGCCGTCA TACTCCGTTC TTCAAAGGCT ACCGTCCGCA GTTCTACTTC 700
CGTACAACTG ACGTGACTGG CACCATCGAA CTGCCGGAAG GCGTTGAGAT 750
GGTAATGCCG GGCGACAACA TCAAAATGGT TGTTACCCTG ATCCACCCGA 800
TCGCGATGGA CGACGG 816
2)INFORMATION FOR SEQ ID N0: 25
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 825 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Citrobacter braakii
(B) STRAIN: ATCC 43162
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 25
CGCGATCCTG GTTGTTGCTG CAACTGACGG CCCGATGCCG CAGACTCGTG 50
AGCACATCCT GCTGGGTCGY CAGGTAGGCG TTCCGTACAT CATCGTGTTC 100
CTGAACAAAT GCGACATGGT TGATGACGAA GAGCTGCTGG AACTGGTAGA 150
AATGGAAGTT CGTGAACTTC TGTCTCAGTA CGATTTCCCG GGCGACGACA 200
CGCCGATCGT TCGTGGTTCT GCTCTGAAAG CGCTGGAAGG CGAWGCAGAG 250
TGGGAAGCGA AAATCATCGA ACTGGCTGGC TTCCTGGATT CTTACATCCC 300
GGAACCAGAG CGTGCGATTG ACAAGCCGTT CCTGCTGCCT ATCGAAGACG 350
TATTCTCCAT CTCTGGTCGT GGTACCGTTG TTACCGGTCG TGTAGAGCGC 400
GGTATCATCA AAGTTGGTGA AGAAGTTGAA ATCGTTGGTA TCAARGACAC 450
TGCTAAGTCT ACCTGTACTG GCGTTGAAAT GTTCCGCAAA CTGCTGGACG 500
AAGGCCGTGC TGGTGAGAAC GTTGGTGTTC TGCTGCGTGG TATCAAGCGT 550
GAAGAAATCG AACGTGGTCA GGTACTGGCT AAGCCGGGCT CTATCAAGCC 600
GCACACCAAG TTCGAATCTG AAGTGTACAT TCTGTCCAAA GACGAAGGCG 650
GCCGTCATAC TCCGTTCTTC AARGGCTACC GTCCGCAGTT CTACTTCCGT 700
ACTACTGACG TGACTGGTAC CATCGAACTG CCGGAAGGCG TTGAGATGGT 750
AATGCCGGGC GACAACATCA AAATGGTTGT TACCCTGATC CACCCAATCG 800
CGATGGACGA CGGTCTGCGT TTCGC 825
2)INFORMATION FOR SEQ ID N0: 26
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 829 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
220
CA 02307010 2000-OS-19
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Citrobacter koseri
(B) STRAIN: ATCC 27156
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 26
CGGCGCGATC CTGGTTGTTG CTGCGACTGA CGGCCCGATG CCGCAGACCC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCGTA CATCATCGTG 100
TTCCTGAACA AATGCGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
TGAGATGGAA GTGCGTGAAC TGCTGTCTCA GTACGATTTC CCGGGCGACG 200
ACACGCCGAT CGTTCGTGGT TCTGCTCTGA AAGCGCTGGA AGGCGAMGCT 250
GAGTGGGAAG CGAAAATCAT CGAACTGGCT GGCTACCTGG ATTCTTACAT 300
CCCGGAACCA GAGCGTGCGA TTGACAAGCC GTTCCTGCTG CCGATCGAAG 350
ACGTATTCTC CATCTCCGGT CGTGGTACCG TTGTTACCGG TCGTGTAGAG 400
CGCGGTATCA TCAAAGTGGG CGAAGAAGTT GAAATYGTTG GTATCAAAGA 450
GACTGCGAAG TCTACCTGTA CTGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGCCG TGCTGGTGAG AACGTAGGTG TTCTGCTGCG TGGTATCAAA 550
CGTGAAGAAA TCGAACGTGG TCAGGTACTG GCTAAGCCGG GYTCCATCAA 600
GCCGCACACC AAGTTCGAAT CTGAAGTGTA CATYCTGTCY AAAGATGAAG 650
GCGGCCGTCA TACTCCGTTC TTCAAAGGCT ACCGTCCGCA GTTCTACTTC 700
CGTACAACTG ACGTGACTGG CACCATCGAA CTGCCGGAAG GCGTAGAGAT 750
GGTAATGCCG GGCGACAACA TCAAAATGGT TGTTACCCTG ATCCACCCGA 800
TCGCGAGGAC GACGGTCTGC GTTTCGCAA 829
2)INFORMATION FOR SEQ ID NO: 27
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 827 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Citrobacter farmeri
(B) STRAIN: ATCC 51112
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 27
CGCGATCCTG GTTGTTGCTG CGACTGACGG CCCGATGCCG CAGACTCGTG 50
AGCACATCCT GCTGGGTCGT CAGGTAGGCG TTCCGTACAT CATCGTGTTC 100
CTGAACAAAT GCGACATGGT TGATGACGAA GAGCTGCTGG AACTGGTAGA 150
GATGGAAGTT CGTGAACTGC TGTCTCAGTA CGATTTCCCG GGCGACGACA 200
CGCCGATCGT TCGTGGTTCT GCTCTGAAAG CGCTGGAAGG CGACGCAGAG 250
TGGGAAGCGA AAATCATCGA ACTGGCAGGC TTCCTGGATT CTTACATCCC 300
GGAACCAGAG CGTGCGATTG ACAAGCCGTT CCTGCTGCCG ATCGAAGACG 350
TATTCTCCAT CTCTGGTCGT GGTACCGTTG TTACCGGTCG TGTAGAGCGC 400
GGTATCATCA AAGTGGGTGA AGAAGTTGAA ATCGTTGGTA TCAAAGAGAC 450
TGCCAAGTCT ACCTGTACTG GCGTTGAAAT GTTCCGCAAA CTGCTGGACG 500
AAGGCCGTGC TGGTGAGAAC GTAGGTGTTC TGCTGCGTGG TATCAAACGT 550
GAAGAAATCG AACGTGGTCA GGTACTGGCT AAGCCGGGCW CCATCAAGCC 600
RCACACTATG TTCGAATCTG AAGTGTACAT TCTGTCCAAA GACGAAGGCG 650
GCCGTCATAC TCCGTTCTTC AAAGGCTACC GTCCGCAGTT CTACTTCCGT 700
ACGACTGACG TGACTGGCAC CATCGAACTG CCGGAAGGTG TTGAGATGGT 750
TATGCCGGGC GACAACATCA AAATGGTTGT TACCCTGATC CACCCGATCG 800
CGATGGACGA CGGTCTGCGT TTCGCAA 827
221
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID N0: 28
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 797 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Citrobacter freundii
(B) STRAIN: ATCC 8090
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 28
CCTGGTTGTT GCTGCGACTG ACGGCCCGAT GCCGCAGACT CGTGAGCACA 50
TCCTGCTGGG TCGTCAGGTA GGCGTTCCGT ACATCATCGT GTTCCTGAAC 100
AAATGCGACA TGGTTGATGA CGAAGAGCTG CTGGAACTGG TAGAAATGGA 150
AGTTCGTGAA CTTCTGTCTC AGTACGATTT CCCGGGCGAC GACACTCCGA 200
TCGTTCGTGG TTCTGCTCTG AAAGCGCTGG AAGGCGAAGC AGAGTGGGAA 250
GCGAAAATCA TCGAACTGGC TGGCTTCCTG GATTCTTACA TCCCAGAACC 300
AGAGCGTGCG ATTGACAAGC CGTTCCTGCT GCCTATCGAA GACGTATTCT 350
CCATCTCCGG TCGTGGTACC GTTGTTACCG GTCGTGTAGA GCGCGGTATC 400
ATCAAAGTTG GTGAAGAAGT TGAAATCGTT GGTATCAAAG AGACTGCTAA 450
GTCTACCTGT ACTGGCGTTG AAATGTTCCG CAAACTGCTG GACGAAGGCC 500
GTGCTGGTGA GAACGTTGGT GTTCTGCTGC GTGGTATCAA ACGTGAAGAA 550
ATCGAACGTG GTCAGGTACT GGCTAAGCCG GGCTCTATCA AGCCGCACAC 600
CAAGTTCGAA TCTGAAGTGT ACATTCTGTC CAAAGACGAA GGCGGCCGTC 650
ATACTCCGTT CTTCAAAGGC TACCGTCCGC AGTTCTACTT CCGTACTACT 700
GACGTGACTG GTACCATCGA ACTGCCGGAA GGCGTAGAGA TGGTAATGCC 750
GGGCGACAAC ATCAAA.ATGG TTGTTACCCT GATCCACCCA ATCGCGA 797
2)INFORMATION FOR SEQ ID N0: 29
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Citrobacter sedlakii
(B) STRAIN: ATCC 51115
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 29
CGGCGCGATC CTGGTTGTTG CCGCGACTGA CGGCCCGATG CCGCAGACCC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCGTA CATCATCGTG 100
TTCCTGAACA AATGCGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
AGAGATGGAA GTTCGTGAAC TGCTGTCTCA GTACGATTTC CCGGGCGACG 200
ACACGCCGAT CGTTCGTGGT TCAGCTCTGA AAGCGCTGGA AGGCGACGCA 250
GAGTGGGAAG CGA.A.AATCATCGAACTGGCT GGCTTCCTGG ATTCTTACAT 300
222
CA 02307010 2000-OS-19
TCCGGAACCA GAGCGTGCGA TTGACAAGCC GTTCCTGCTG CCGATCGAAG 350
ACGTATTCTC CATCTCCGGT CGTGGTACCG TTGTTACCGG TCGTGTAGAG 400
CGCGGTATCA TCAAAGTGGG CGAAGAAGTT GAAATCGTTG GTATCAAAGA 450
GACTGCGAAG TCTACCTGTA CTGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGCCG TGCGGGTGAG AACGTAGGTG TTCTGCTGCG TGGTATCAAA 550
CGTGAAGAAA TCGAACGTGG TCAGGTACTG GCGAAGCCGG GCACCATCAA 600
GCCGCACACC AAGTTCGAAT CTGAAGTGTA TATTCTGTCC AAAGATGAAG 650
GCGGCCGTCA TACTCCGTTC TTCAAAGGCT ACCGTCCGCA GTTCTACTTC 700
CGTACAACTG ACGTGACTGG CACCATCGAA CTGCCGGAAG GCGTAGAGAT 750
GGTAATGCCG GGCGACAACA TCAAAATGGT TGTTACCCTG ATCCACCCGA 800
TCGCGATGGA CGACGGTCTG CGTTTC 826
2)INFORMATION FOR SEQ ID NO: 30
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 823 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Citrobacter werkmanii
(B) STRAIN: ATCC 51114
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 30
GCGATCCTGG TTGTTGCTGC GACTGACGGC CCGATGCCGC AGACTCGTGA 50
GCACATCCTG CTGGGTCGTC AGGTAGGCGT TCCGTACATC ATCGTGTTCC 100
TGAACAAATG CGACATGGTT GATGACGAAG AGCTGCTGGA ACTGGTAGAA 150
ATGGAAGTTC GTGAACTTCT GTCTCAGTAC GATTTCCCGG GCGACGACAC 200
TCCGATCGTT CGTGGTTCTG CTCTGAAAGC GCTGGAAGGC GAAGCAGAGT 250
GGGAAGCGAA AATCATCGAA CTGGCTGGCT TTCTGGATTC TTACATCCCG 300
GAACCAGAGC GTGCGATTGA CAAGCCGTTC CTGCTRCCTA TCGAAGACGT 350
ATTCTCCATC TCCGGTCGTG GTACCGTTGT TACCGGTCGT GTAGAGCGCG 400
GTATCATCAA AGTTGGTGAA GAAGTTGAAA TCGTTGGTAT CAAAGACACC 450
GCTAAGTCTA CCTGTACCGG CGTTGAAATG TTCCGCAAAC TGCTGGACGA 500
AGGCCGTGCT GGTGAGAACG TTGGTGTTCT GCTGCGTGGT ATCAAACGTG 550
AAGAAATCGA ACGTGGTCAG GTACTGGCTA AGCCGGGCTC TATCAAGCCG 600
CACACCAAGT TCGAATCTGA AGTGTACATC CTGTCCAAAG ACGAAGGCGG 650
CCGTCATACT CCGTTCTTCA AAGGCTACCG TCCGCAGTTC TACTTCCGTA 700
CTACTGACGT GACTGGTACC ATCGAACTGC CGGAAGGCGT AGAGATGGTA 750
ATGCCGGGCG ACAACATYAA AATGGTTGTT ACYCTGATCC ACCCGATCGC 800
GATGGACGAC GGTCTGCGTT TCG 823
2)INFORMATION FOR SEQ ID NO: 31
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
223
CA 02307010 2000-OS-19
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Citrobacter youngae
(B) STRAIN: ATCC 29935
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 31
GGCGCGATCC TGGTTGTTGC TGCGACTGAC GGCCCGATGC CGCAGACTCG 50
TGAGCACATC CTGCTGGGTC GTCAGGTAGG CGTTCCGTAC ATCATCGTGT 100
TCCTGAACAA ATGCGACATG GTTGATGACG AAGAGCTGCT GGAACTGGTA 150
GAAATGGAAG TTCGTGAACT TCTGTCTCAG TACGATTTCC CGGGCGACGA 200
TACGCCGATC GTTCGTGGTT CTGCTCTGAA AGCGCTGGAA GGCGAAGCAG 250
AGTGGGAAGC GAAAATCATC GAACTGGCTG GCTTCCTGGA TTCTTACATC 300
CCGGAACCAG AACGTGCTAT CGATAAGCCG TTCCTGCTGC CAATCGAAGA 350
CGTATTCTCC ATCTCCGGTC GTGGTACCGT TGTTACTGGT CGTGTAGAAC 400
GCGGTATCAT CAAAGTTGGT GAAGAAGTTG AAATCGTTGG TATCAAAGAG 450
ACTGCCAAGT CTACCTGTAC TGGCGTTGAA ATGTTCCGCA AACTGCTGGA 500
CGAAGGCCGT GCTGGTGAGA ACGTTGGTGT TCTGCTGCGT GGTATCAAAC 550
GTGAAGAAAT CGAACGTGGT CAGGTACTGG CTAAGCCGGG CTCTATCAAG 600
CCGCACACCA AGTTCGAATC TGAAGTGTAC ATTCTGTCCA AAGACGAAGG 650
CGGCCGTCAT ACTCCGTTCT TCAAAGGCTA CCGTCCGCAG TTCTACTTCC 700
GTACTACTGA CGTGACGGGT ACCATCGAAC TGCCGGAAGG CGTAGAGATG 750
GTAATGCCGG GCGACAACAT CAAAATGGTT GTTACCCTGA TCCACCCAAT 800
CGCGATGGAT GACGGTCTGC GTTTCG 826
2)INFORMATION FOR SEQ ID N0: 32
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 841 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Clostridium perfringens
(B) STRAIN: ATCC 13124
(xi)SEQUENCE DESCRIPTION: SEQ ID N0:32
CGGAGCTATA TTAGTTTGTT CAGCAGCTGA TGGTCCAATG CCTCAAACAA 50
GAGAGCACAT CTTATTATCA TCAAGAGTTG GAGTTGACCA CATCGTAGTA 100
TTCTTAAACA AAGCAGATAT GGTTGACGAC GAAGAATTAT TAGAATTAGT 150
TGAAATGGAA GTTAGAGAGT TATTAAGCGA GTACAACTTC CCAGGAGACG 200
AYATTCCAGT AATCAARGGA TCAGCTTTAG TAGCATTAGA AAACCCAACT 250
GACGAAGCTG CAACAGCTTG TATCAGAGAG TTAATGGATG CTGTAGATAG 300
CTACATCCCA ACACCAGAAA GAGCAACAGA TAAGCCATTC TTAATGCCAG 350
TAGAGGACGT ATTCACAATC ACTGGTAGAG GAACAGTTGC AACAGGAAGA 400
GTTGAAAGAG GAGTTCTACA TGTAGGAGAC GAAGTAGAAG TAATCGGATT 450
AACTGAAGAA AGAAGAAAA.ACTGTTGTAAC AGGAATCGAA ATGTTCAGAA 500
AGTTATTAGA TGAAGCACAA GCTGGAGATA ACATCGGAGC ATTATTAAGA 550
GGTATCCAAA GAACTGAYAT CGAAAGAGGT CAAGTTTTAG CTCAAGTTGG 600
AACAATCAAC CCACACAAAA AATTCGTAGG TCAAGTATAC GTACTTAAAA 650
AAGAAGAAGG TGGAAGACAT ACTCCATTCT TCGATGGATA CAGACCACAA 700
TTCTACTTCA GAACAACAGA CGTTACAGGA TCAATCAAAT TACCAGAAGG 750
AATGGAAATG GTTATGCCTG GAGACCACAT CGACATGGAA GTTGAATTAA 800
TCACAGAAAT CGCTATGGAY GAAGGATTAA GATTCGCTAT C 841
224
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID NO: 33
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 822 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Comamonas acidovorans
(B) STRAIN: ATCC 15668
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 33
CGGCGCCATC CTGGTGTGCT CGGCCGCTGA CGGCCCCATG CCCCAGACCC 50
GCGAGCACAT CCTGCTGGCC CGTCAGGTGG GCGTGCCCTA CATCATCGTG 100
TTCCTGAACA AGTGCGACAT GGTGGACGAC GAAGAGCTGC TGGAACTGGT 150
CGAAATGGAA GTGCGCGAGC TGCTTGCCAA GTACGACTTC CCCGGCGACG 200
ACACCCCCAT CATCCGCGGC TCGGCCAAGC TGGCCCTGGA AGGCGACCAG 250
TCCGACAAGG GCGAACCTGC CATCCTGCGC CTGGCTGAAG CACTGGACTC 300
CTACATCCCC ACGCCCGAGC GCGCTGTGGA CGGCGCCTTT GCAATGCCCG 350
TGGAAGACGT GTTCTCGATC TCTGGCCGTG GCACCGTGGT GACTGGCCGT 400
ATCGAGCGCG GCATCATCAA GGTCGGCGAA GAAATCGAAA TCGTCGGTAT 450
CCGCGACACC CAGAAGACCA TCGTCACCGG CGTGGAAATG TTCCGCAAGC 500
TGCTGGACCA AGGTCAAGCT GGCGACAACG TGGGTCTGCT GCTGCGCGGC 550
ACCAAGCGTG AAGACGTGGA ACGCGGCCAA GTGCTGTGCA AGCCCGGCTC 600
CATCAAGCCC CACACCCACT TCACGGCTGA GGTGTACGTG CTGTCCAAGG 650
ACGAAGGTGG TCGCCACACT CCGTTCTTCA ACAACTACCG TCCCCAGTTC 700
TATTTCCGTA CGACCGACGT GACCGGCTCC ATCGAGCTGC CCGCCGACAA 750
GGAAATGGTG ATGCCTGGCG ACAACGTGTC GATCACCGTC AAGCTGATCG 800
CCCCCATCGC CATGGAAGAA GG 822
2)INFORMATION FOR SEQ ID NO: 34
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 702 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium bovis
(B) STRAIN: ATCC 7715
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 34
GCCGCAGACC CGTGAGCACG TCCTCCTGGC CCGTCAGGTC GGTGTGCCCT 50
ACATCCTCGT CGCCCTCAAC AAGTGCGACA TGGTCGACGA CGAGGACCTC 100
ATCGAGCTCG TCGAGATGGA GGTCCGTGAG CTCCTCGCCG AGCAGGACTA 150
CGACGAGGAC GCCCCGATCA TCCACATCTC CGCCCTCAAG GCCCTCGAGG 200
GTGACCCGGA GTGGACGCAG CGCATCGTCG ACCTCATGAA GGCCTGCGAC 250
GACGCCATCC CGGATCCGGA GCGCGAGACG GACAAGCCGT TCCTCATGCC 300
225
CA 02307010 2000-OS-19
GATCGAGGAC ATCTTCACGA TCACCGGCCG CGGCACCGTC GTCACGGGCC 350
GTGTCGAGCG TGGCATCCTC AACGTCAACG AGGAGGTCGA GATCCTGGGT 400
ATCTGCGAGA ACTCCCAGAA GACGACCGTC ACCTCCATCG AGATGTTCAA 450
CAAGTTCCTC GACACGGCCG AGGCCGGCGA CAACGCCGCC CTGCTGCTCC 500
GTGGCCTGAA GCGCGAGGAC GTCGAGCGTG GCCAGATCGT GGCCAAGCCG 550
GGCGCCTACA CGCCGCACAC CGAGTTCGAG GGCTCCGTGT ACATCCTCTC 600
CAAGGACGAG GGTGGCCGCC ACACGCCGTT CTTCGACAAC TACCGTCCGC 650
AGTTCTACTT CCGGACGACC GACGTCACCG GCGTCGTCAA GCTGCCGGAG 700
GG 702
2)INFORMATION FOR SEQ ID N0: 35
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 689 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium cervicis
(B) STRAIN: NCTC 10604
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 35
GGCTCAGACC CGCGAGCACG TTCTGCTTGC TCGCCAGGTT GGCGTTCCGA 50
CGATCCTGGT TGCCCTCAAC AAGGCCGATA TGGTCGACGA TGAGGAAATG 100
CTGGAGCTCG TTGAGGAAGA GTGCCGCGAC CTGCTCGAGT CCCAGGACTT 150
CGATCGTGAC GCCCCGATCA TCCAGGTTTC CGCGCTGAAG GCTCTCGAAG 200
GTGATCCGCA GTGGGTTGCT AAGGTCGAGG AGCTCATGGA GGCAGTCGAC 250
ACCTTCGTGC CGACTCCTGA GCGCGACATG GACAAGCCGT TCCTCATGCC 300
GATCGAAGAC GTCTTCACCA TCACCGGCCG TGGCACCGTT GTTACCGGTC 350
GTGTTGAGCG TGGCAAGCTC CCGATCAACT CTGAGGTTGA AATCCTCGGT 400
ATCCGCGAAC CGCAGAAGAC CACCGTTACC GGTATCGAGA TGTTCCACAA 450
GTCCATGGAT GAAGCATGGG CAGGCGAGAA CTGTGGTCTC CTCCTGCGTG 500
GCACCAAGCG CGATGAGGTT GAGCGCGGTC AGGTCGTTGC CGTTCCCGGT 550
TCGATCACCC CGCACACCAA CTTCACCGGA CAGGTCTACA TCCTCAAGAA 600
GGAAGAAGGC GGTCGTCACA ACCCGTTCTT CTCGAACTAC CGTCCGCAGT 650
TCTACTTCCG CACCACGGAC GTGACCGGCG TCATCACCC 689
2)INFORMATION FOR SEQ ID N0: 36
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium flavescens
(B) STRAIN: ATCC 10340
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 36
226
CA 02307010 2000-OS-19
GGTTGTTGCT GCAACCGATG GTCCTATGCC GCAGACCCGC GAGCACGTTC 50
TTCTGGCTCG CCAGGTTGGC GTTCCTTACA TCCTCGTTGC TCTTAACAAG 100
TGCGACATGG TTGATGATGA GGAAATCATC GAGCTCGTTG AGATGGAAAT 150
CCGCGAACTG CTCGCTGAGC AGGACTACGA CGAGGATGCC CCCATCATCC 200
ACATCTCCGC TCTCAAGGCT CTTGAGGGTG ACGAGAAGTG GGTACAGGCC 250
ATCGTCGACC TCATGCAGGC CTGCGATGAC TCCATTCCGG ATCCGGAGCG 300
CGAGACCGAC AAGCCCTTCC TCATGCCTAT CGAGGACATC TTCACCATCA 350
CCGGCCGCGG TACCGTTGTT ACCGGCCGTG TTGAGCGTGG CGTTTTGAAG 400
GTCAACGAGG ATGTTGAGAT CATCGGCATC AAGGAGAAGT CCATCTCCAC 450
CACCGTTACC GGTATCGAAA TGTTCCGCAA GATGATGGAC TACACCGAGG 500
CTGGCGACAA CTGTGGTCTG CTTCTGCGTG GTACCAAGCG TGAAGAGGTC 550
GAGCGCGGCC AGGTTGTTAT CAAGCCGGGC GCCTACACCC CCCACACCAA 600
GTTCGAGGGT TCCGTCTACG TCCTCAAGAA GGAAGAGGGC GGCCGCCACA 650
CCCCGTTCAT GGACAACTAC CGTCCGCAGT TCTACTTCCG TACCACTGAC 700
GTGACCGGCG TTGTTCACCT GCCTGAGGGC ACCGAGATGG TCATGCCTGG 750
CGACAACGTT GATATGACCG TTGAGCTCAT CCAGCCCGTC GCTAGGATGA 800
GGGC 804
2)INFORMATION FOR SEQ ID NO: 37
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 692 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium kutscheri
(B) STRAIN: ATCC 15677
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 37
TGCCTCAGAC CCGTGAGCAC GTTCTTCTTG CTCGCCAGGT TGGCGTTCCT 50
TACATCCTCG TTGCTCTTAA CAAGTGCGAC ATGGTTGACG ATGAGGAAAT 100
CATCGAGCTC GTTGAGATGG AAGTTCGCGA GCTTCTTGCT GAGCAGGAGT 150
ACGATGAAGA GGCTCCAATC ATCCACATCT CTGCTTTGAA GGCTCTTGAG 200
GGCGACGAGA AGTGGACTCA GGCCATCATC GACCTCATGC AGGCTTGTGA 250
TGACTCCATC CCAGATCCAG AGCGTGAGAC CGACAAGCCA TTCCTCATGC 300
CTATCGAGGA TATCTTCACC ATCACCGGTC GTGGCACCGT TGTTACCGGT 350
CGTGTTGAGC GCGGTTCCTT GAAGGTGAAT GAGGACGTCG AGATCATCGG 400
CATCAAGGAG AAGTCCACCA CTACTACCGT TACCGGTATC GAAATGTTCC 450
GTAAGCTTCT TGATTACACC GAAGCTGGCG ATAACTGTGG TCTGCTTCTT 500
CGTGGTATCA AGCGCGAAGA CGTTGAGCGT GGTCAGGTTG TTGTTAAGCC 550
AGGCGCTTAC ACACCTCACA CCGAGTTCGA GGGCTCTGTT TACGTTCTTT 600
CCAAGGACGA GGGCGGCCGC CACACCCCAT TCTTCGACAA CTACCGTCCA 650
CAGTTCTACT TCCGCACCAC TGACGTTACC GGTGTTGTGA AG 692
2)INFORMATION FOR SEQ ID N0: 38
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 797 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
227
CA 02307010 2000-OS-19
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium minutissimum
(B) STRAIN: ATCC 23348
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 38
CCTGGTTGTT GCTGCAACCG ATGGCCCGAT GCCGCAGACC CGCGAGCACG 50
TTCTTCTGGC CCGCCAGGTT GGCGTTCCGT ACATCCTCGT TGCACTGAAC 100
AAGTGTGACA TGGTTGACGA TGAGGAAATC ATCGAGCTCG TTGAGATGGA 150
GATCCGTGAG CTGCTCGCTG AGCAGGACTA CGACGAGGAA GCTCCGATCG 200
TTCACATCTC CGCTCTGAAG GCTCTTGAGG GCGACGAGAA GTGGGCACAG 250
TCCATCGTTG ACCTGATGCA GGCTTGCGAT GACTCCATCC CGGATCCGGA 300
GCGCGAGCTG GACAAGCCGT TCCTGATGCC GATCGAGGAC ATCTTCACCA 350
TTACCGGCCG CGGTACCGTT GTTACCGGCC GTGTTGAGCG TGGCTCCCTG 400
AACGTTAACG AGGACATCGA GATCATCGGT ATCAAGGACA AGTCCATGTC 450
CACCACCGTT ACCGGTATCG AGATGTTCCG CAAGATGATG GACTACACCG 500
AGGCTGGCGA CAACTGTGGT CTGCTTCTGC GTGGTACCAA GCGTGAAGAG 550
GTTGAGCGTG GCCAGGTTTG CATCAAGCCG GGCGCTTACA CCCCGCACAC 600
CAAGTTCGAG GGTTCCGTCT ACGTCCTGAA GAAGGAAGAG GGCGGCCGCC 650
ACACCCCGTT CATGGACAAC TACCGTCCGC AGTTCTACTT CCGCACCACC 700
GACGTCACCG GTGTCATCAA GCTGCCGGAG GGCACCGAGA TGGTCATGCC 750
GGGCGACAAC GTTGAGATGT CCGTAGAGCT GATCCAGCCG GTCGCTA 797
2)INFORMATION FOR SEQ ID N0: 39
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 702 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium mycetoides
(B) STRAIN: ATCC 21134
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 39
GCCGCAGACC CGCGAGCACG TTCTTCTGGC CCGCCAGGTC GGCGTCCCCT 50
ACATCCTCGT TGCGCTGAAC AAGTGCGACA TGGTTGATGA TGAGGAGATC 100
ATCGAGCTCG TGGAGATGGA GGTCCGTGAG CTGCTCGGCG AGCAGGACTA 150
CGACGAGGAC GCCCCCATCA TCCACATCTC CGCTCTGAAG GCTCTCGAGG 200
GCGACGAGAA GTGGGTTCAG TCCGTGCTCG ACCTCATGCA GGCGTGCGAC 250
GACTCCATCC CGGATCCGGT CCGCGAGACC GACCGCGACT TCCTGATGCC 300
GATCGAGGAC ATCTTCACCA TCTCCGGCCG CGGCACCGTG GTTACCGGTC 350
GTGTGGAGCG CGGCGTGCTC AACCTCAACG ACGAGGTCGA GATCATCGGC 400
ATCCGCGACA AGTCCCAGAA GACCACCGTC ACCTCCATCG AGATGTTCAA 450
CAAGCTGCTC GATACCGCTG AGGCAGGCGA CAACGCGGCT CTGCTGCTCC 500
GCGGTCTGAA GCGCGAGGAC GTCGAGCGTG GCCAGGTTGT CATCAAGCCG 550
GGCGCCTACA CCCCGCACAC CAAGTTCGAG GGTTCCGTCT ACGTCCTGTC 600
CAAGGACGAG GGCGGCCGCC ACACCCCGTT CTTCGACAAC TACCGTCCGC 650
AGTTCTACTT CCGCACCACC GACGTGACCG GTGTTGTGAA GCTGCCGGAG 700
GG 702
228
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID N0: 40
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 674 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium pseudogenitalium
(B) STRAIN: ATCC 33038
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 40
GCTCGCCAGG TTGGCGTTCC TTACATCCTC GTTGCGCTGA ACAAGTGCGA 50
CATGGTTGAT GATGAGGAAA TCATCGAGCT CGTTGAGATG GAGATCCGTG 100
AGCTGCTCGC AGAGCAGGAT TACGATGAGG AAGCTCCTAT CGTTCACATC 150
TCCGCTCTGA AGGCCCTCGA GGGCGATGAC AAGTGGGTAC AGTCCGTCGT 200
TGATCTGATG GAAGCCTGCG ACAACTCCAT CCCGGATCCG GAGCGCGCTA 250
CCGACCAGCC GTTCCTGATG CCTATCGAGG ACATCTTCAC CATTACCGGC 300
CGCGGTACCG TTGTTACCGG CCGTGTTGAG CGTGGCCGTC TGAACGTCAA 350
CGAGGACGTT GAGATCATCG GTATCCAGGA GAAGTCCCAG ACCACCACCG 400
TTACCGGTAT CGAGATGTTC CGCAAGATGA TGGACTACAC CGAGGCTGGC 450
GACAACTGTG GTCTGCTTCT GCGTGGTACC AAGCGTGAGG ACGTTGAGCG 500
TGGCCAGGTT GTTATCAAGC CGGGCGCTTA CACCCCGCAC ACCAAGTTCG 550
AGGGCTCCGT CTACGTCCTG AAGAAGGAAG AGGGCGGCCG CCACACCCCG 600
TTCATGAACA ACTACCGTCC GCAGTTCTAC TTCCGTACCA CGGACGTTAC 650
CGGTGTTGTT CACCTGCCAG AGGG 674
2)INFORMATION FOR SEQ ID NO: 41
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 694 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium renale
(B) STRAIN: ATCC 19412
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 41
TGCCTCAGAC CCGTGAGCAC GTTCTGCTTG CTCGTCAGGT CGGCGTTCCT 50
TACATCCTCG TTGCACTGAA CAAGTGCGAC ATGGTCGACG ACGAAGAAAT 100
CATCGAGCTC GTCGAGATGG AAATCCGTGA ACTGCTCGCA GAGCAGGACT 150
ACGATGAGGA AGCTCCTATC GTTCACATCT CCGCTCTGGG CGCCCTGAAC 200
GGCGAGCAGA AGTGGGTTGA CTCCATCGTC GAACTGATGG AAGCTTGCGA 250
CAACTCCATC CCAGACCCAG TTCGCGACAT CGACCACCCA TTCCTGATGC 300
CTATCGAGGA CATCTTCACC ATTACCGGTC GCGGTACCGT TGTTACCGGC 350
CGTGTCGAGC GTGGCCGTCT CAACGTCAAC GAAGAAGTTG AGATCATCGG 400
229
CA 02307010 2000-OS-19
TATCAAGGAC AAGTCCCAGA AGACCACCGT CACCGGTATC GAGATGTTCC 450
GCAAGATGCT GGACTACACC GAAGCTGGCG ACAACTGTGG TCTGCTGCTC 500
CGCGGCATCG GCCGTGAGGA TGTCGAGCGT GGCCAGGTTA TCATCAAGCC 550
AGGCGCTTAC ACCCCTCACT CTGAGTTCGA GGGCTCTGTC TACGTCCTGT 600
CCAAGGACGA GGGTGGCCGC CACACCCCAT TCTTCGACAA CTACCGTCCA 650
CAGTTCTACT TCCGCACCAC CGACGTGACC GGCGTTGTGC ACCT 694
2)INFORMATION FOR SEQ ID NO: 42
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 687 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium ulcerans
(B) STRAIN: NCTC 8665
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 42
GCCGCAGACC CGCGAGCACG TTCTGCTGGC TCGCCAGGTT GGCGTTCCKT 50
ACATCCTSGT TGCACTGAAC AAGTGCGACA TGGTTGACGA TGAGGARCTC 100
CTSGAGCTCG TCGAGATGGA GGTCCGCGAG CTGCTGGCTG AGCAGGACTA 150
CGACGAGGAA GCTCCGRTCG TTCACATCTC CGCWCTGAAC GCCCTGGACG 200
GCGACSAGAA GTGGGCTVAC TCCATCCTCG AGCTGATGCA GGCTTGCGAC 250
GAGTCCATCC CGGATCCGGA GCGCGAGACC GACAAGCCGT TCCTGATGCC 300
GATTGAGGAC ATCTTCACCA TTACCGGTCG CGGYACCGTT GTTACCGGCC 350
GTGTTGAGCG TGGCDTCCTG AACGTSAACG ACGASGTTGA GATCATGGGY 400
ATCCGGGAGA AGTCCCAGAA GACCACCGTY ACCKSCATCG AGATGTTCAA 450
CAAGMTGMTG GACWCCGCAG AGGCTGGCGA CAACGCTGSW CTGCTGCTGC 500
GTGGTMTSAA GCGTGAGGAC GTTGAGCGTG GCCAGATCAT CGYTAAGCCG 550
GGCGCKTACA CCCCGCACAC CGAGTTCGAG GGCTCCGTCT ACGTCCTGTC 600
CAAGGACGAG GGCGGCCGCC ACACCCCGTT CTTCGACAAC TACCGTCCGC 650
AGTTCTACTT CCGCACCACC GACGTSACCG GTGTTGT 687
2)INFORMATION FOR SEQ ID N0: 43
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 778 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium urealyticum
(B) STRAIN: ATCC 43042
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 43
CTGGTTGTTG CTGCAACCGA TGGCCCGATG CCGCAGACCC GTGAGCACGT 50
TCTGCTGGCT CGCCAGGTTG GCGTTCCGTA CATCCTCGTT GCACTGAACA 100
230
CA 02307010 2000-OS-19
AGTGCGACAT GGTTGACGAT GAGGAGCTCC TCGAGCTCGT CGAGATGGAG 150
GTCCGCGAGC TTCTGGCTGA GCAGGACTAC GACGAGGAGG CTCCGGTCGT 200
CCCGATCTCC GCACTGGGCG CCCTGGACGG CGATCAGAAG TGGGTCGACT 250
CCATCCTCGA GCTCATGAAG GCTTGCGACG AGTCCATCCC GGACCCGGAG 300
CGCGAGACCG ACAAGCCGTT CCTGATGCCG GTTGAGGACA TCTTCACCAT 350
TACCGGTCGC GGCACCGTCG TTACCGGCCG TGTTGAGCGT GGCGTCCTGA 400
ACCTGAACGA CGAGGTCGAG ATCCTGGGCA TCCGCGAGAA GTCCACCAAG 450
ACCACCGTCA CCTCCATCGA GATGTTCAAC AAGCTGCTGG ACACCGCAGA 500
GGCTGGCGAC AACGCTGCAC TGCTGCTGCG TGGTCTGAAG CGTGAGGACG 550
TCGAGCGAGG CCAGATCATC GCTAAGCCGG GCGCTTACAC CCCGCACACC 600
GAGTTCGAGG GCTCCGTCTA CGTCCTGTCC AAGGACGAGG GCGGCCGTCA 650
CACCCCGTTC TTCGACAACT ACCGTCCGCA GTTCTACTTC CGTACCACCG 700
ACGTCACCGG TGTCGTTACC CTGCCAGAGG GCACCGACAT GGTCATGCCG 750
GGCGACAACG TTGAGATGAG CGTCAAGC 778
2)INFORMATION FOR SEQ ID N0: 44
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 703 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium xerosis
(B) STRAIN: ATCC 373
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 44
CGCAGACCCG TGAGCACGTC CTCCTGGCCC GCCAGGTCGG CGTCCCCTAC 50
ATCCTCGTCG CCCTGAACAA GTGCGACATG GTCGACGATG AGGAGATCAT 100
CGAGCTCGTG GAGATGGAGG TGCGTGAGCT TCTCGCCGAG CAGGACTACG 150
ACGAGGAGGC CCCGATCGTG CACATCTCCG CCCTGGGCGC CCTCAATGGC 200
GAAGAGAAGT GGGTCGACTC CATCGTCGAG CTCATGAACG CCGTCGACGA 250
GAACGTTCCG GACCCGGTCC GCGAGACCGA CAAGCCGTTC CTGATGCCCG 300
TCGAGGACAT CTTCACCATC ACCGGCCGCG GCACCGTCGC CACCGGTCGC 350
GTGGAGCGCG GCACCCTGAA GGTCAACGAC GAGGTCGAGA TCCTGGGCAT 400
CCAGGAGAAG TCCCAGACCA CCACCGTCAC CGGCATCGAG ATGTTCCGCA 450
AGCTGCTGGA CTCCGCCGAG GCCGGCGACA ACTGTGGCCT GCTGCTCCGC 500
GGCATCAAGC GCGAGGACAT CGAGCGCGGC CAGATCATCG CGAAGCCGGG 550
CGCCTACACC CCGCACACCG AGTTCGAGGG CTCCGTCTAC ATCCTGGCCA 600
AGGACGAGGG CGGCCGCCAC ACCCCGTTCT TCGACAACTA CCGTCCGCAG 650
TTCTACTTCC GCACCACCGA CGTCACCGGC GTCGTGAAGC TGCCGGAGGG 700
CAC 703
2)INFORMATION FOR SEQ ID NO: 45
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 832 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
231
CA 02307010 2000-OS-19
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Coxiella burnetii
(B) STRAIN: Nine Mile phase II
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 45
GGAGCGATAT TGGTGGTGAG CGCAGCGGAC GGCCCGATGC CGCAAACGCG 50
GGAACACATT GTATTGGCGA AGCAAGTGGG TGTTCCGAAC ATAGTGGTTT 100
ACTTGAACAA AGCGGACATG GTGGATGACA AAGAGCTGTT GGAATTAGTG 150
GAAATGGAAG TGAGGGATTT ATTGAACAGT TATGATTTCC CTGGGGATGA 200
GACGCCGATA ATAGTGGGGT CAGCGTTAAA GGCGTTAGAA GGTGACAAGA 250
GTGAGGTTGG GGAGCCATCG ATAATCAAAT TAGTGGAAAC GATGGACACG 300
TACTTCCCGC AGCCGGAGCG AGCGATAGAC AAACCGTTTT TAATGCCGAT 350
CGAAGATGTG TTTTCGATAT CGGGCCGAGG GACGGTGGTG ACGGGACGCG 400
TAGAGCGAGG GATCATCAAA GTGGGCGACG AGATAGAGAT TGTGGGGATC 450
AAGGACACGA CGAAGACGAC GTGCACGGGC GTTGAGATGT TTCGCAAATT 500
ATTGGATGAA GGTCAAGCGG GTGACAACGT AGGAATTTTA TTGAGAGGGA 550
CGAAACGCGA AGAAGTGGAG CGTGGTCAAG TATTGGCGAA ACCGGGATCG 600
ATCACGCCAC ACAAGAAATT TGAGGCGGAG ATTTATGTGT TGTCGAAGGA 650
AGAAGGGGGA CGCCACACAC CGTTTTTACA AGGCTATCGA CCGCAATTTT 700
ATTTCCGCAC GACGGACGTG ACGGGCCAGT TATTGAGTTT ACCGGAGGGG 750
ATAGAGATGG TGATGCCGGG AGATAACGTG AAAGTGACGG TTGAATTGAT 800
TGCGCCGGTA GCGATGGATG AAGGGCTACG AT 832
2)INFORMATION FOR SEQ ID N0: 46
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 816 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Edwardsiella hoshinae
(B) STRAIN: ATCC 33379
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 46
GGCGCTATCC TGGTTGTTGC TGCGACTGAC GGCCCGATGC CGCAGACCCG 50
TGAGCACATC CTGCTGGGTC GCCAGGTAGG CGTTCCGTAC ATCATCGTGT 100
TCCTGAACAA GTGCGACATG GTTGATGACG AAGAGCTGCT GGAACTGGTT 150
GAGATGGAAG TTCGCGAACT GCTGTCTCAG TACGATTTCC CGGGCGACGA 200
TACGCCGGTA ATCCGCGGTT CTGCGCTGAA AGCGCTGGAA GGCGAAGCCG 250
AGTGGGAAGC GAAGATCATC GAACTGGCTG AAACGCTGGA CTCCTACATT 300
CCGGAACCTG AGCGTGACAT CGACAAGCCG TTCCTGCTGC CGATCGAAGA 350
CGTATTCTCA ATCTCTGGTC GTGGTACCGT TGTTACCGGT CGTGTAGAGC 400
GCGGTATCAT CAAGGTAGGC GACGAAGTTG AAATCGTAGG TATCAAGCCG 450
ACCACCAAGA CTACCTGTAC TGGCGTTGAA ATGTTCCGCA AACTGCTGGA 500
CGAAGGCCGT GCTGGTGAGA ACGTAGGTGT TCTGCTGCGT GGTACCAAGC 550
GTGACGAAAT CGAACGTGGT CAGGTACTGG CTAAGCCGGG CACCATCACT 600
CCGCACACCA AGTTCGAATC AGAAGTGTAC ATCCTGAGCA AGGATGAAGG 650
CGGCCGTCAT ACTCCGTTCT TCAAAGGTTA CCGTCCGCAG TTCTACTTCC 700
GTACCACTGA CGTGACTGGC ACCATCGAAC TGCCGGAAGG CGTAGAGATG 750
GTAATGCCGG GCGACAACAT CAAGATGGTT GTTACCCTGA TCCACCCGAT 800
CGCCATGGAC GATGGT 816
232
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID N0: 47
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 821 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Edwardsiella tarda
(B) STRAIN: ATCC 15947
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 47
GGCGCGATCC TGGTTGTTGC TGCGACTGAC GGCCCGATGC CGCAGACCCG 50
TGAGCACATC CTGTTGGGTC GCCAGGTAGG CGTTCCGTAC ATCATCGTGT 100
TCCTGAACAA GTGCGACATG GTTGATGACG AAGAGCTGCT GGAACTGGTT 150
GAGATGGAAG TTCGCGAACT GCTGTCTCAG TACGACTTCC CGGGCGACGA 200
CACGCCGGTA ATCCGCGGTT CTGCGCTGAA AGCGCTGGAA GGCGAAGCCG 250
AGTGGGAAGC GAAGATCATC GAACTGGCTG AAACTCTGGA CTCCTACATC 300
CCGGAACCTG AGCGTGACAT CGACAAGCCG TTCCTGCTGC CGATCGAAGA 350
CGTATTCTCT ATCTCTGGCC GTGGTACCGT TGTTACCGGT CGTGTAGAGC 400
GCGGTATCAT CAAGGTAGGC GACGAAGTTG AAATCGTTGG TATCAAGCCG 450
ACCACCAAGA CCACCTGTAC TGGCGTTGAA ATGTTCCGCA AACTGCTGGA 500
CGAAGGCCGT GCTGGTGAGA ACGTTGGTGT TCTGCTGCGT GGTACTAAGC 550
GTGACGAAAT CGAACGTGGT CAGGTACTGG CTAAGCCGGG CACCATCACT 600
CCGCACACCA AGTTCGAATC TGAAGTGTAC ATCCTGAGCA AGGATGAAGG 650
CGGCCGTCAT ACTCCGTTCT TCAAAGGCTA CCGTCCGCAG TTCTACTTCC 700
GTACTACTGA CGTGACTGGT ACCATCGAAC TGCCGGAAGG CGTAGAGATG 750
GTAATGCCGG GCGACAACAT CAAGATGGTT GTTACCCTGA TCCACCCGAT 800
CGCCATGGAC GATGGTCTGC G 821
2)INFORMATION FOR SEQ ID N0: 48
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 830 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Eikenella corrodens
(B) STRAIN: ATCC 23834
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 48
CGGTGCCATC CTGGTGGTAT CCGCTGCTGA CGGCCCCATG CCTCAGACTC 50
GCGAACACAT CCTGTTGGCT CGTCAGGTAG GTGTACCCTA CATCCTCGTA 100
TTCATGAACA AATGCGACAT GGTAGATGAT GCCGAGCTGC TTGAGTTGGT 150
TGAGATGGAA ATCCGCGACC TGCTCTCCAG CTATGACTTC CCTGGTGACG 200
ACTGCCCGAT CGTACAAGGT TCCGCTCTCA AAGCCCTCGA AGGCGATGCC 250
233
CA 02307010 2000-OS-19
GGTTACAAAG AAAAAATCTT CGAACTAGCT GCTGCTTTGG ATAGCTACAT 300
CCCCACTCCT CAACGTGCTG TAGACAAACC CTTCCTGTTG CCGATCGAAG 350
ACGTATTCTC TATCTCCGGC CGTGGTACCG TAGTAACCGG TCGTGTAGAG 400
CGCGGCATCA TCAAAGTAGG TGAAGAGATC GAAATCGTTG GTCTGAAGCC 450
CACTCAGAAA ACTACCTGTA CTGGCGTGGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGTCA GGCCGGTGAC AACGTAGGCG TACTGCTGCG CGGTACCAAA 550
CGTGAAGAAG TTGAGCGTGG TCAAGTATTG GCTAAACCCG GCACCATCAC 600
TCCGCACACC AAGTTCAAAG CCGAAGTATA CGTATTGAGC AAAGAAGAAG 650
GTGGTCGTCA CACCCCGTTC TTTGCCAACT ACCGTCCACA GTTCTACTTC 700
CGTACTACTG ACGTAACCGG TGCTGTAGAG CTGGAGCCTG GTGTAGAAAT 750
GGTTATGCCT GGTGAGAACG TAACCATCAC CGTAGAACTG ATTGCTCCGA 800
TTGCTATGGA AGAAGGTCTG CGCTTTGCGA 830
2)INFORMATION FOR SEQ ID NO: 49
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80$ bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterobacter aerogenes
(B) STRAIN: ATCC 13048
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 49
GGCGCGATCC TGGTTGTTGC TGCGACTGAC GGCCCGATGC CGCAGACTCG 50
TGAGCACATC CTGCTGGGTC GTCAGGTAGG CGTTCCGTAC ATCATCGTGT 100
TCCTGAACAA ATGCGACATG GTTGATGACG AAGAGCTGCT GGAACTGGTT 150
GAGATGGAAG TTCGTGAACT GCTGTCTCAG TACGATTTCC CGGGCGACGA 200
CACTCCGATC GTTCGTGGTT CTGCTCTGAA AGCGCTGGAA GGCGACGCAG 250
AGTGGGAAGC GAAAATCATC GAACTGGCTG GCTTCCTGGA TTCTTACATC 300
CCRGAACCAG AGCGTGCGAT TGACAAGCCG TTCCTGCTGC CGATCGAAGA 350
CGTATTCTCC ATCTCCGGTC GTGGTACCGT TGTTACCGGT CGTGTAGAGC 400
GCGGTATCAT CAAAGTTGGT GAAGAAGTTG AAATCGTTGG TATCAAAGAC 450
ACCGCGAAAA CCACCTGTAC TGGCGTTGAA ATGTTCCGCA AACTGCTGGA 500
CGAAGGCCGT GCTGGTGAGA ACGTAGGYGT TCTGCTGCGT GGTATCAAAC 550
GTGAAGAAAT CGAACGTGGT CAGGTACTGG CTAAGCCGGG CAGCATCAAG 600
CCGCACACCA AGTTCGAATC TGAAGTGTAC ATCCTGTCCA AAGACGAAGG 650
CGGCCGTCAT ACTCCGTTCT TCAAAGGCTA CCGTCCGCAG TTCTACTTCC 700
GTACTACTGA CGTGACTGGT ACCATCGAAC TGCCGGAAGG CGTAGAGRTG 750
GTAATGCCGG GCGACAACAT CAAAATGGTT GTTACCCTGA TCCACCCGAT 800
CGCGATGG 808
2)INFORMATION FOR SEQ ID NO: 50
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 828 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
234
CA 02307010 2000-OS-19
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterobacter agglomerans
(B) STRAIN: ATCC 27989
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 50
CGGCGCGATC CTGGTTGTTG CTGCGACTGA CGGCCCGATG CCGCAGACTC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCGTA CATCATCGTG 100
TTCCTGAACA AATGTGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
TGAAATGGAA GTTCGTGAAC TTCTGTCTCA GTACGATTTC CCGGGCGACG 200
ATACTCCGAT CGTTCGTGGT TCTGCTCTGA AAGCGCTGGA AGGCGAMGCW 250
GAGTGGGAAG CGAA.AATCATCGARCTGGCT GGCCACCTGG ATACCTATAT 300
CCCGGAACCA GAGCGTGCGA TTGACAAGCC GTTCCTGCTG CCGATCGAAG 350
ACGTATTCTC CATCTCCGGT CGCGGTACCG TTGTTACCGG TCGTGTAGAG 400
CGCGGTATCA TYAAAGTGGG CGAAGAAGTT GAAATCGTTG GTATCAAAGA 450
TACYGCGAAA TCAACCTGTA CCGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGCCG TGCTGGTGAG AACGTTGGTG TTCTGCTGCG TGGTATCAAA 550
CGTGAAGAAA TCGAACGTGG TCAGGTACTG GCTAAGCCGG GCACCATCAA 600
GCCGCACACC AAGTTCGAAT CTGAAGTGTA CATTCTGTCC AAAGATGAAG 650
GCGGTCGTCA CACTCCGTTC TTCAAAGGCT ACCGTCCSCA GTTCTACTTC 700
CGTACAACTG ACGTGACTGG CACCATCGAA CTGCCGGAAG GCGTAGAGAT 750
GGTAATGCCG GGCGACAACA TCAAAATGGT TGTTACCCTG ATCCACCCGA 800
TCGCGATGGA CGACGGTCTG CGTTCGCA 828
2)INFORMATION FOR SEQ ID NO: 51
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 825 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterobacter amnigenus
(B) STRAIN: ATCC 33072
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 51
TGGCGCGATC CTGGTTGTTG CTGCAACTGA TGGCCCTATG CCACAGACGC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCTTA CATCATCGTG 100
TTCCTGAACA AATGCGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
AGAAATGGAA GTTCGTGAAC TTCTGTCTCA GTACGATTTC CCAGGTGATG 200
ACACTCCAAT CATCCGTGGT TCTGCTCTGA AAGCGCTGGA AGGCGAAGCA 250
GAGTGGGAAG CTAA.AATCGTTGAGCTGGCT GGCTACCTGG ATTCTTACAT 300
CCCGGAACCA GAACGTGCTA TCGATAAGCC ATTCCTGCTG CCAATCGAAG 350
ACGTATTCTC TATCTCCGGC CGTGGTACTG TTGTAACCGG TCGTGTAGAG 400
CGCGGTATCG TTAAAGTTGG CGAAGAAGTT GAAATCGTTG GTATCAAAGA 450
GACTGCTAAG TCTACCTGTA CTGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGCCG TGCTGGTGAG AACGTTGGTG TTCTGCTGCG TGGTATCAAA 550
CGTGAAGAAA TCGAACGTGG TCAGGTACTG GCTAAGCCAG GCTCAATCAA 600
GCCGCACACC AAATTCGAAT CTGAAGTTTA TATTCTGTCC AAAGATGAAG 650
GCGGCCGTCA TACTCCGTTC TTCAAAGGCT ACCGTCCACA GTTCTACTTC 700
CGTACAACTG ACGTGACCGG CACCATCGAA CTGCCAGAAG GCGTAGAGAT 750
GGTAATGCCA GGCGACAACA TTCAGATGGT TGTTACCCTG ATCCACCCAA 800
TCGCGATGGA TGACGGTCTG CGTTT 825
235
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID NO: 52
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 822 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterobacter asburiae
(B) STRAIN: ATCC 35953
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 52
CGGCGCGATC CTGGTTGTTG CTGCGACTGA CGGCCCAATG CCTCAGACTC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCTTT CATCATCGTG 100
TTCCTGAACA AATGCGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
AGAGATGGAA GTTCGTGAAC TGCTGTCTCA GTACGATTTC CCGGGCGACG 200
ATACTCCAAT CGTTCGTGGT TCTGCTCTGA AAGCGCTGGA AGGCGACGCA 250
GAGTGGGAAG CGAAAATCAT CGAACTGGCT GGCTTCCTGG ATTCTTACAT 300
CCCAGAACCA GAGCGTGCGA TTGACAAGCC ATTCCTGCTG CCAATCGAAG 350
ACGTATTCTC CATCTCCGGT CGTGGTACCG TTGTTACCGG TCGTGTAGAG 400
CGCGGTATCA TCAAAGTTGG CGAAGAAGTT GAAATCGTTG GTATCAAAGA 450
GACTGCTAAG TCTACCTGTA CTGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGCCG TGCTGGTGAG AACGTTGGTG TTCTGCTGCG TGGTATCAAA 550
CGTGAAGAAA TCGAACGTGG TCAGGTTCTG GCGAAGCCAG GCTCAATCAA 600
GCCACACACC AAGTTCGAAT CTGAAGTGTA CATCCTGTCC AAAGACGAAG 650
GCGGCCGTCA TACTCCGTTC TTCAAAGGCT ACCGTCCACA GTTCTACTTC 700
CGTACAACTG ACGTGACCGG TACCATCGAA CTGCCAGAAG GCGTTGAGAT 750
GGTAATGCCA GGCGACAACA TCAAGATGGT TGTGACTCTG ATCCACCCAA 800
TCGCGATGGA CGACGGTCTG CG 822
2)INFORMATION FOR SEQ ID N0: 53
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterobacter cancerogenus
(B) STRAIN: ATCC 35317
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 53
CGGCGCGATC CTGGTTGTTG CTGCGACTGA CGGCCCAATG CCTCAGACTC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCTTA CATCATCGTG 100
TTCCTGAACA AGTGCGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
AGAAATGGAA GTTCGTGAAC TGCTGTCTCA GTACGATTTC CCAGGCGACG 200
ACACTCCAAT CGTTCGTGGT TCCGCGCTGA AAGCGCTGGA AGGCGAAGCT 250
236
CA 02307010 2000-OS-19
GAGTGGGAAG CAA.A.AATCATCGAACTGGCT GGCTTCCTGG ATTCTTACAT 300
CCCAGAACCA GAGCGTGCGA TTGACAAGCC ATTCCTGCTG CCAATCGAAG 350
ACGTATTCTC CATCTCCGGT CGTGGTACCG TTGTTACCGG TCGTGTAGAG 400
CGCGGTATCA TCAAAGTTGG TGAAGAAGTT GAAATCGTTG GTATCAAAGA 450
TACTGCKAAA TCTACCTGTA CTGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGCCG TGCTGGTGAG AACGTTGGTG TTCTGCTGCG TGGTATCAAA 550
CGCGAAGAAA TCGAACGTGG TCAGGTTCTG GCGAAGCCAG GCTCAATCAA 600
GCCACACACC AAGTTCGAAT CTGAAGTGTA CATCCTGTCC AAAGACGAAG 650
GCGGCCGTCA TACTCCGTTC TTCAAAGGCT ACCGTCCACA GTTCTACTTC 700
CGTACAACTG ACGTGACCGG TACCATCGAA CTGCCAGAAG GCGTAGAGAT 750
GGTAATGCCA GGCGACAACA TCAAGATGGT TGTGACGCTG ATCCACCCAA 800
TCGCGATGGA CGACGGTCTG CGTTTC 826
2)INFORMATION FOR SEQ ID N0: 54
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 806 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterobacter cloacae
(B) STRAIN: ATCC 13047
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 54
GATCCTGGTA GTAGCTGCGA CTGACGGCCC AATGCCTCAG ACTCGTGAGC 50
ACATCCTGCT GGGTCGTCAG GTAGGCGTTC CTTACATCAT CGTGTTCCTG 100
AACAAATGCG ACATGGTTGA TGACGAAGAG CTGCTGGAAC TGGTAGAGAT 150
GGAAGTTCGT GAACTGCTGT CTCAGTACGA TTTCCCAGGC GACGATACCC 200
CAATCGTTCG TGGTTCTGCT CTGAAAGCGC TGGAAGGCGA CGCAGAGTGG 250
GAAGMGAAAA TCATCGAACT GGCTGGCTAC CTGGATTCTT ACATCCCAGA 300
ACCAGAGCGT GCGATTGAYA AGCCATTCCT GCTGCCAATC GAAGACGTAT 350
TCTCCATCTC CGGTCGTGGT ACCGTTGTTA CCGGTCGTGT AGAGCGCGGT 400
ATCATCAAAG TGGGTGAAGA AGTTGAAATC GTTGGTATCA AAGAGACTGC 450
GAAGTCTACC TGTACTGGCG TTGAAATGTT CCGCAAACTG CTGGACGAAG 500
GCCGTGCTGG TGAGAACGTT GGTGTTCTGC TGCGTGGTAT CAAACGTGAA 550
GAAATCGAAC GTGGTCAGGT TCTGGCGAAG CCAGGCTCAA TCAAGCCACA 600
CACCAAGTTC GAATCTGAAG TGTACATCCT GTCCAAAGAC GAAGGCGGCC 650
GTCATACTCC GTTCTTCAAA GGCTACCGTC CACAGTTCTA CTTCCGTACA 700
ACTGACGTGA CCGGTACCAT CGAACTGCCA GAAGGCGTAG AGGTGGTAAT 750
GCCAGGCGAC AACATCAAGA TGGTTGTGAC TCTGATCCAC CCAATCGCGA 800
TGGACG 806
2)INFORMATION FOR SEQ ID NO: 55
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
237
CA 02307010 2000-OS-19
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterobacter gergoviae
(B) STRAIN: ATCC 33028
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 55
CGGCGCGATC CTGGTTGTTG CTGCGACTGA CGGCCCGATG CCGCAGACCC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCGTA CATCATCGTG 100
TTCCTGAACA AGTGCGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
AGAGATGGAA GTTCGTGAAC TGCTGTCTCA GTACGATTTC CCGGGCGACG 200
ACACCCCGAT CGTTCGCGGT TCTGCGCTGA AAGCGCTGGA AGGCGACGCA 250
GAGTGGGAAG CGAAAATCAT CGAACTGGCT GGCCACCTGG ATACCTAYAT 300
CCCGGAACCA GAGCGTGCGA TTGACAAGCC GTTCCTGCTG CCGATCGAAG 350
ACGTATTCTC CATTTCCGGT CGTGGTACCG TTGTTACCGG TCGTGTAGAG 400
CGCGGTATCA TCAAGGTTGG TGAAGAAGTT GAAATCGTTG GTATCAAAGA 450
CACCGCGAAA ACCACCTGTA CTGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGCCG TGCTGGTGAG AACGTCGGCG TTCTGCTGCG TGGTATCAAG 550
CGTGAAGAAA TCGAACGTGG TCAGGTACTG GCTAAGCCGG GCTCCATCAA 600
GCCGCACACC AAGTTCGAAT CTGAAGTGTA CATCCTGTCC AAAGACGAAG 650
GCGGCCGTCA CACTCCGTTC TTCAAAGGCT ACCGTCCGCA GTTCTACTTC 700
CGTACAACTG ACGTGACTGG CACCATCGAA CTGCCGGAAG GCGTAGAGAT 750
GGTAATGCCG GGCGACAACA TCAAGATGGT TGTTACCCTG ATCCACCCGA 800
TCGCGATGGA CGACGGTCTG CGTTTC 826
2)INFORMATION FOR SEQ ID NO: 56
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 829 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterobacter hormaechei
(B) STRAIN: ATCC 49162
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 56
GGCGCGATCC TGGTTGTTGC TGCGACTGAC GGCCCTATGC CTCAGACCCG 50
TGAGCACATC CTGCTGGGTC GTCAGGTAGG CGTTCCTTAC ATCATCGTGT 100
TCCTGAACAA ATGCGACATG GTTGATGACG AAGAGCTGCT GGAACTGGTA 150
GAGATGGAAG TTCGTGAACT GCTGTCTCAG TACGATTTCC CAGGCGACGA 200
CACCCCAATC GTTCGTGGTT CCGCGCTGAA AGCGCTGGAA GGCGAMGCAG 250
AGTGGGAAGM GAAAATCATC GARCTGGCTG GCTTCCTGGA TTCTTACATC 300
CCAGAACCAG AGCGTGCGAT TGACAAGCCA TTCCTGCTGC CAATCGAAGA 350
CGTATTCTCC ATCTCCGGTC GTGGTACCGT TGTTACCGGT CGTGTWGAGC 400
GCGGTATCAT CAAAGTAGGT GAAGAAGTTG AAATCGTTGG TATCAAAGAG 450
ACTGCGAAGT CTACCTGTAC TGGCGTTGAA ATGTTCCGCA AACTGCTGGA 500
CGAAGGCCGT GCTGGTGAGA ACGTTGGTGT TCTGCTGCGT GGTATCAAAC 550
GTGAAGAAAT CGAACGTGGT CAGGTTCTGG CGAAGCCAGG CTCAATCAAG 600
CCACACACCA AGTTCGAATC TGAAGTGTAC ATTCTGTCCA AAGACGAAGG 650
CGGCCGTCAT ACTCCGTTCT TCAAAGGCTA CCGTCCACAG TTCTACTTCC 700
GTACAACTGA CGTGACCGGT ACCATCGAAC TGCCAGAAGG CGTAGAGATG 750
GTAATGCCAG GCGACAACAT CAAGATGGTT GTGACGCTGA TCCACCCAAT 800
CGCGATGGAC GACGGTCTGC GTTTCGCAA 829
238
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID NO: 57
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 831 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterobacter sakazakii
(B) STRAIN: ATCC 29544
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 57
GGCGCTATCC TGGTTGTTGC TGCGACTGAC GGCCCGATGC CGCAGACCCG 50
TGAGCACATC CTGCTGGGTC GTCAGGTAGG CGTTCCGTAC ATCATCGTGT 100
TCCTGAACAA ATGCGACATG GTTGATGACG AAGAGCTGCT GGAACTGGTT 150
GAGATGGAAG TGCGCGAGCT GCTGTCTCAG TACGACTTCC CGGGCGACGA 200
CACCCCGATC GTTCGTGGTT CTGCTCTGAA AGCGCTGGAA GGCGACGCTG 250
AGTGGGAAGC GAAAATCATC GAGCTGGCAG GTCACCTGGA TTCCTACATC 300
CCGGAACCGG AGCGTGCGAT TGACAAGCCG TTCCTGCTGC CGATCGAAGA 350
CGTATTCTCC ATCTCYGGTC GTGGTACCGT TGTTACCGGT CGTGTAGAGC 400
GCGGTATCAT CAAGGTTGGT GAAGAAGTTG AAATCGTGGG CATCAAAGAC 450
ACCGCGAAAT CCACCTGTAC CGGCGTTGAA ATGTTCCGCA AACTGCTGGA 500
CGAAGGCCGT GCGGGCGAGA ACGTAGGTGT TCTGCTGCGT GGTATCAAAC 550
GTGAAGAAAT CGAACGTGGT CAGGTACTGG CTAAGCCGGG CTCCATCAAG 600
CCGCACACCA AGTTCGAATC TGAAGTGTAC ATTCTGTCCA AAGATGAAGG 650
CGGCCGTCAC ACTCCGTTCT TCAAAGGCTA CCGTCCGCAG TTCTACTTCC 700
GTACRACTGA CGTGACTGGC ACCATCGAAC TGCCGGAAGG CGTTGAGATG 750
GTAATGCCGG GCGACAACAT CAAAATGGTT GTTACCCTGA TCCACCCGAT 800
CGCGATGGAC GACGGTCTGC GTTTCGCAAT C 831
2)INFORMATION FOR SEQ ID N0: 58
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus casseliflavus
(B) STRAIN: ATCC 2578$
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 58
CGGCGCGATC TTAGTAGTAT CTGCTGCTGA TGGTCCTATG CCTCAAACAC 50
GTGAACACAT CTTGTTATCA CGTAACGTTG GTGTACCATA CATCGTTGTT 100
TTCTTAAACA AAATGGATAT GGTTGATGAC GAAGAATTAC TAGAATTAGT 150
TGAAATGGAA GTTCGTGACT TATTGTCAGA ATATGACTTC CCAGGCGACG 200
ATGTTCCTGT AATCGCTGGT TCTGCTTTGA AAGCTCYTGA AGGCGATGCT 250
239
CA 02307010 2000-OS-19
TCATACGAAG AAAAAATCAT GGAATTAATG GCTGCAGTTG ACGAATACGT 300
TCCAACTCCA GAACGTGACA CTGACAAACC ATTCATGATG CCAGTCGAAG 350
ACGTATTCTC AATCACTGGA CGTGGTACTG TTGCTACAGG CCGTGTTGAA 400
CGTGGACAAG TTCGCGTTGG TGACGAAGTT GAAATCGTTG GTATTGCTGA 450
AGAAACTGCT AAAACAACTG TAACTGGTGT TGAAATGTTC CGTAAATTGT 500
TAGACTATGC TGAAGCAGGG GATAACATTG GTGCATTGCT ACGTGGTGTT 550
GCTCGTGAAG ACATCCAACG TGGACAAGTA TTGGCTAAAG CTGGTACAAT 600
CACACCTCAT ACAA.AATTTAAAGCTGAAGT TTACGTTTTA ACAAAAGAAG 650
AAGGTGGACG TCACACACCA TTCTTCACTA ACTACCGTCC TCAGTTCTAC 700
TTCCGTACAA CTGACGTAAC TGGTGTTGTT GAATTACCAG AAGGAACTGA 750
AATGGTTATG CCTGGTGATA ACGTAACAAT CGACGTTGAA TTGATCCACC 800
CAATCGCTAT CGAAGACGGA ACTCGTTTCT CAATT 835
2)INFORMATION FOR SEQ ID N0: 59
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus cecorum
(B) STRAIN: ATCC 43198
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 59
GGTGCTATCT TAGTAGTATC TGCTGCTGAT GGTCCTATGC CACAAACTCG 50
TGAACACATT CTTTTATCAC GTAACGTTGG TGTTCCATAC ATCGTTGTTT 100
TCTTAAACAA AGTTGATATG GTTGACGACG AAGAATTATT AGAATTAGTT 150
GAAATGGAAG TACGTGACTT ATTAACTGAA TACGACTTCC CAGGAGACGA 200
TGTTCCTGTA ATCGCTGGTT CTGCATTAAA AGCTTTAGAA GGCGACCCAT 250
CTTACGAAGA AAAAATCTTA GAATTAATGG CTGCAGTTGA CGAATACATC 300
CCAACTCCAG AACGTGACAA CGATAAACCA TTCATGATGC CAGTCGAAGA 350
CGTATTTTCA ATCACTGGTC GTGGTACTGT TGCTACAGGT CGTGTTGAAC 400
GTGGACAAGT ACGTGTTGGT GACGAAGTTG AAATAGTTGG TATCCATGAT 450
GAAATTTCTA AAACAACAGT TACTGGTGTT GAAATGTTCC GTAAATTATT 500
AGATTACGCT GAAGCTGGAG ACAACATCGG TGCATTATTA CGTGGTGTGG 550
CTCGTGAAGA TATCCAACGT GGTCAAGTAT TAGCTAAACC AGGTTCAATC 600
ACTCCACATA CAAAATTCAC TGCTGAAGTG TACGTTTTAA CTAAAGAAGA 650
AGGTGGACGT CATACTCCAT TCTTCACTAA CTACCGTCCA CAATTCTACT 700
TCCGTACAAC TGACGTTACA GGTGTAGTTA ACTTACCAGA AGGTACTGAA 750
ATGGTTATGC CTGGTGATAA CGTAACTATG GAAGTTGAAT TAATCCACCC 800
AATCGCTATC GAAGACGGAA CTCGTT 826
2)INFORMATION FOR SEQ ID NO: 60
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
240
CA 02307010 2000-OS-19
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus dispar
(B) STRAIN: ATCC 51266
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 60
CGGCGCGATC TTGGTAGTAT CTGCTGCTGA TGGTCCTATG CCTCAAACTC 50
GTGAACACAT CCTATTGTCA CGTAACGTTG GTGTTCCTTA CATCGTCGTT 100
TTCTTGAACA AAATGGACAT GGTTGATGAC GAAGAATTAT TAGAATTAGT 150
TGAAATGGAA GTTCGTGACT TATTGTCAGA ATACGACTTC CCAGGCGACG 200
ACACTCCAGT TATCGCAGGT TCAGCTTTGA AAGCCTTAGA AGGCGACGCT 250
TCATATGAAG AAAAAATCTT AGAATTAATG GCTGCAGTTG ACGAATATAT 300
CCCAACTCCA GTTCGTGATA CTGACAAACC ATTCATGATG CCAGTCGAAG 350
ATGTATTCTC AATCACTGGT CGTGGTACTG TTGCAACTGG TCGTGTTGAA 400
CGTGGACAAG TTCGCGTTGG TGACGAAGTT GAAATCGTAG GTATCGCTGA 450
AGAAACTGCT AAAACTACTG TAACAGGTGT TGAAATGTTC CGTAAATTGT 500
TGGATTACGC TGAAGCTGGC GACAACATTG GTGCATTATT ACGTGGTGTG 550
GCTCGTGAAG ATATCCAACG TGGTCAAGTA TTATCAAAAC CAGGTTCAAT 600
CACTCCACAT ACAAAATTTG CGGCAGAAGT TTACGTTTTA ACTAAAGAAG 650
AAGGTGGACG TCATACTCCA TTCTTCACTA ACTACCGCCC ACAATTCTAC 700
TTCCGTACAA CTGACGTAAC AGGTGTTGTT GAATTACCAG AAGGTACTGA 750
AATGGTTATG CCTGGCGATA ACGTTACTAT GGACGTTGAA TTAATCCACC 800
CAATCGCGAT CGAAGACGGT ACTCGTTTCT CAATC 835
2)INFORMATION FOR SEQ ID NO: 61
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus durans
(B) STRAIN: ATCC 19432
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 61
CGGAGCTATC TTAGTAGTTT CTGCTGCTGA TGGCCCTATG CCTCAAACTC 50
GTGAACATAT CCTATTATCT CGTCAAGTTG GTGTTCCTTA CATCGTYGTA 100
TTCTTGAACA AAGTAGATAT GGTCGATGAC GAAGAATTAC TAGAATTAGT 150
TGAAATGGAA GTTCGTGACT TATTAACAGA ATACGAATTC CCTGGTGACG 200
ATGTTCCTGT AATCGCTGGT TCAGCTTTGA AAGCTTTAGA AGGCGACGCT 250
TCATACGAAG AAA.A.AATCCTTGAATTAATG GCTGCAGTTG ACGAATATAT 300
CCCAACTCCA GAACGTGACA ACGACAAACC ATTCATGATG CCAGTTGAAG 350
ATGTATTCTC RATCACTGGT CGTGGTACTG TTGCTACAGG TCGTGTTGAA 400
CGTGGACAAG TTCGCGTTGG TGACGTTGTA GATATCGTTG GTATCGCAGA 450
AGAAACAGCT CAAACAACAG TTACTGGTGT TGAAATGTTC CGTAAATTAT 500
TAGRCTACGC TGAAGCTGGA GACAACATTG GTGCTTTACT ACGTGGTGTT 550
GCACGTGAAG ACATCCAACG TGGACAAGTT TTAGCTAAAC CAGGTACAAT 600
CACKCCTCAT ACAAAATTCT CTGCAGAAGT ATACGTGTTG ACTAAAGAAG 650
AAGGTGGACG TCATACTCCA TTCTTCACTA ACTACCGTCC ACAATTCTAC 700
TTCCGTACAA CTGACGTAAC AGGTGTTGTT GAATTACCAG AAGGAACTGA 750
AATGGTTATG CCTGGCGACA ACGTAACAAT GGAAGTTGAA TTAATCCACC 800
CAATCGCTAT CGAAAATGGT ACTAAATTCT CAATC 835
241
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID N0: 62
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 680 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus faecalis
(B) STRAIN: 8610
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 62
AGTAGTTTCT GCTGCTGATG GTCCTATGCC TCAAACACGT GAACATATCT 50
TATTATCACG TAACGTTGGT GTACCATACA TCGTTGTATT CTTAAACAAA 100
ATGGATATGG TTGATGACGA AGAATTATTA GAATTAGTAG AAATGGAAGT 150
TCGTGACTTA TTATCAGAAT ACGATTTCCC AGGCGATGAT GTTCCAGTTA 200
TCGCAGGTTC TGCTTTGAAA GCTTTAGAAG GCGACGAGTC TTATGAAGAA 250
AAAATCTTAG AATTAATGGC TGCAGTTGAC GAATATATCC CAACTCCAGA 300
ACGTGATACT GACAAACCAT TCATGATGCC AGTCGAAGAC GTATTCTCAA 350
TCACTGGACG TGGTACTGTT GCTACAGGCC GTGTTGAACG TGGTGAAGTT 400
CGCGTTGGTG ACGAAGTTGA AATCGTTGGT ATTAAAGACG AAACATCTAA 450
AACAACTGTT ACAGGTGTTG AAATGTTCCG TAAATTATTA GACTACGCTG 500
AAGCAGGCGA CAACATCGGT GCTTTATTAC GTGGTGTAGC ACGTGAAGAT 550
ATCGAACGTG GACAAGTATT AGCTAAACCA GCTACAATCA CTCCACACAC 600
AAAATTCAAA GCTGAAGTAT ACGTATTATC AAAAGAAGAA GGCGGACGTC 650
ACACTCCATT CTTCACTAAC TACCGTCCTC 680
2)INFORMATION FOR SEQ ID N0: 63
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 680 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus faecalis
(B) STRAIN: 8487
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 63
AGTAGTTTCT GCTGCTGATG GTCCTATGCC TCAAACACGT GAACATATCT 50
TATTATCACG TAACGTTGGT GTACCATACA TCGTTGTATT CTTAAACAAA 100
ATGGATATGG TTGATGACGA AGAATTATTA GAATTAGTAG AAATGGAAGT 150
TCGTGACTTA TTATCAGAAT ACGATTTCCC AGGCGATGAT GTTCCAGTTA 200
TCGCAGGTTC TGCTTTGAAA GCTTTAGAAG GCGACGAGTC TTATGAAGAA 250
AAAATCTTAG AATTAATGGC TGCAGTTGAC GAATATATCC CAACTCCAGA 300
ACGTGATACT GACAAACCAT TCATGATGCC AGTCGAAGAC GTATTCTCAA 350
TCACTGGACG TGGTACTGTT GCTACAGGCC GTGTTGAACG TGGTGAAGTT 400
242
CA 02307010 2000-OS-19
CGCGTTGGTG ACGAAGTTGA AATCGTTGGT ATTAAAGACG AAACATCTAA 450
AACAACTGTT ACAGGTGTTG AAATGTTCCG TAAATTATTA GACTACGCTG 500
AAGCAGGCGA CAACATCGGT GCTTTATTAC GTGGTGTAGC ACGTGAAGAT 550
ATCGAACGTG GACAAGTATT AGCTAAACCA GCTACAATCA CTCCACACAC 600
AAAATTCAAA GCTGAAGTAT ACGTATTATC AAAAGAAGAA GGCGGACGTC 650
ACACTCCATT CTTCACTAAC TACCGTCCTC 680
2)INFORMATION FOR SEQ ID N0: 64
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 685 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus faecium
(B) STRAIN: 8482
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 64
AGTAGTTTCT GCTGCTGACG GCCCAATGCC TCAAACTCGT GAACACATCC 50
TATTGTCTCG TCAAGTTGGT GTTCCTTACA TCGTTGTATT CTTGAACAAA 100
GTAGACATGG TTGATGACGA AGAATTACTA GAATTAGTTG AAATGGAAGT 150
TCGTGACCTA TTAACAGAAT ACGAATTCCC TGGTGACGAT GTTCCTGTAG 200
TTGCTGGATC AGCTTTGAAA GCTCTAGAAG GCGACGCTTC ATACGAAGAA 250
AAAATTCTTG AATTAATGGC TGCAGTTGAC GAATACATCC CAACTCCAGA 300
ACGTGACAAC GACAAACCAT TCATGATGCC AGTTGAAGAC GTGTTCTCAA 350
TTACTGGACG TGGTACTGTT GCTACAGGTC GTGTTGAACG TGGACAAGTT 400
CGCGTTGGTG ACGAAGTTGA AGTTGTTGGT ATTGCTGAAG AAACTTCAAA 450
AACAACAGTT ACTGGTGTTG AAATGTTCCG TAAATTGTTA GACTACGCTG 500
AAGCTGGAGA CAACATTGGT GCTTTACTAC GTGGTGTTGC ACGTGAAGAC 550
ATCCAACGTG GACAAGTTTT AGCTAAACCA GGTACAATCA CACCTCATAC 600
AAAATTCTCT GCAGAAGTAT ACGTGTTGAC AAAAGAAGAA GGTGGACGTC 650
ATACTCCATT CTTCACTAAC TACCGTCCTC AATTT 685
2)INFORMATION FOR SEQ ID NO: 65
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 825 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus flavescens
(B) STRAIN: ATCC 49996
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 65
CGGCGCGATC TTAGTAGTAT CTGCTGCTGA TGGTCCTAYG CCTCAAACAC 50
GTGAACACAT CTTGTTATCA CGTAACGTTG GTGTACCATA CATCGTTGTT 100
243
CA 02307010 2000-OS-19
TTCTTAAACA AAATGGATAT GGTTGATGAC GAAGAATTAC TAGAATTAGT 150
TGAAATGGAA GTTCGTGACT TATTGTCAGA ATATGACTTC CCAGGCGACG 200
ATGTTCCTGT AATCGCTGGT TCTGCTTTGA AAGCTCTTGA AGGCGATGCT 250
TCATACGAAG AA.A.A.AATCATGGAATTAATG GCTGCAGTTG ACGAATACGT 300
TCCAACTCCA GAACGTGACA CTGACAAACC ATTCATGATG CCAGTCGAAG 350
ACGTATTCTC AATCACTGGA CGTGGTACTG TTGCTACAGG CCGTGTTGAA 400
CGTGGACAAG TTCGCGTTGG TGACGAAGTT GAAATCGTTG GTATTGCTGA 450
AGAAACTGCT AAAACAACTG TAACTGGTGT TGAAATGTTC CGTAAATTGT 500
TAGACTATGC TGAAGCAGGG GATAACATTG GTGCATTGCT ACGTGGGGTT 550
GCTCGTGAAG ACATCCAACG TGGACAAGTA TTAGCTAAAG CTGGTACAAT 600
CACACCTCAT ACAA.A.ATTTAAAGCTGAAGT TTACGTTTTA ACAAAAGAAG 650
AAGGTGGACG TCACACTCCA TTCTTCACTA ACTACCGTCC TCAGTTCTAC 700
TTCCGTACAA CTGACGTAAC TGGTGTTGTT GAATTACCAG AAGGAACTGA 750
AATGGTTATG CCTGGTGATA AMGTAACAAT CGACGTTGAA TTGATCCACC 800
CAATCGCTAT CGAAGACGGA ACTCG 825
2)INFORMATION FOR SEQ ID N0: 66
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 636 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus gallinarum
(B) STRAIN: 8420
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 66
TCCTATGCCT CAAACTCGTG AACACATCTT GTTATCACGT AACGTTGGCG 50
TACCATACAT CGTTGTTTTC TTGAACAAAA TGGATATGGT TGATGACGAA 100
GAATTGCTAG AATTAGTTGA AATGGAAGTT CGTGACCTAT TGTCTGAGTA 150
TGACTTCCCA GGCGACGATG TTCCTGTAAT CGCCGGTTCT GCTTTGAAAG 200
CTCTTGAAGG AGATCCTTCA TACGAAGAAA AAATCATGGA ATTGATGGCT 250
GCAGTTGACG AATACGTTCC AACTCCAGAA CGTGATACTG ACAAACCATT 300
CATGATGCCA GTCGAAGACG TATTCTCAAT CACTGGACGT GGTACTGTTG 350
CTACAGGCCG TGTTGAACGT GGACAAGTTC GCGTTGGTGA TGAAGTAGAA 400
ATCGTTGGTA TTGCTGACGA AACTGCTAAA ACAACTGTAA CAGGTGTTGA 450
AATGTTCCGT AAATTGTTAG ACTATGCTGA AGCAGGGGAT AACATTGGTG 500
CATTGCTACG TGGGGTTGCT CGTGAAGACA TCCAACGTGG ACAAGTATTG 550
GCTAAAGCTG GTACAATCAC ACCTCATACA AAATTCAAAG CTGAAGTTTA 600
TGTTTTGACA AAAGAAGAAG GTGGACGTCA CACTCC 636
2)INFORMATION FOR SEQ ID N0: 67
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
244
CA 02307010 2000-OS-19
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus hirae
(B) STRAIN: ATCC 8043
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 67
CGGAGCTATC TTAGTAGTTT CTGCTGCTGA TGGTCCTATG CCTCAAACTC 50
GTGAACATAT CCTAYTATCT CGTCAAGTTG GTGTTCCATA CATCGTTGTA 100
TTCTTGAACA AAGTAGATAT GGTTGACGAC GAAGAATTAC TAGAATTAGT 150
TGAAATGGAA GTTCGTGACT TATTAACAGA ATACGAATTC CCTGGTGACG 200
ATGTTCCTGT AGTTGCTGGT YCAGCTTTGA AAGCTTTAGA AGGCGACGCT 250
TCATACGAAG AAAAA.ATCCTTGAATTGATG GCTGCAGTTG ACGAATATAT 300
CCCAACTCCA GAACGTGACA ACGACAAACC ATTCATGATG CCAGTCGAAG 350
ACGTATTCTC AATCACTGGT CGTGGTACTG TTGCTACAGG TCGTGTTGAA 400
CGTGGACAAG TTCGCGTTGG TGACGTTGTA GATATCGTTG GTATCGCAGA 450
AGAAACAGCT CAAACAACAG TTACTGGTGT TGAAATGTTC CGTAAATTAT 500
TAGACTACGC TGAAGCTGGA GACAACATTG GTGCTTTACT ACGTGGTGTT 550
GCACGTGAAG ACATCCAACG TGGACAAGTT TTAGCTAAAC CAGGTACAAT 600
CACACCTCAT ACAAAATTCT CTGCAGAAGT ATACGTGTTG ACAA.AAGAAG 650
AAGGTGGACG TCATACTCCA TTCTTCACTA ACTACCGTCC ACAATTCTAC 700
TTCCGTACRA CTGACGTAAC AGGTGTTGTT GAATTACCAG AAGGAACTGA 750
AATGGTTATG CCTGGCGACA ACGTAACAAT GGAAGTTGAA TTAATCCACC 800
CAATCGCTAT CGAAAACGGT ACTAAATTCT CAATC 835
2)INFORMATION FOR SEQ ID NO: 68
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus mundtii
(B) STRAIN: ATCC 43186
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 68
CGGAGCAATC TTAGTTGTTT CTGCTGCTGA CGGCCCTATG CCTCAAACTC 50
GTGAACACAT CCTATTATCT CGTCAAGTTG GTGTACCATA CATCGTTGTA 100
TTCTTGAACA AAGTAGATAT GGTTGATGAC GAAGAATTAC TTGAATTAGT 150
TGAAATGGAA GTTCGTGACC TATTAACAGA ATACGAATTC CCTGGTGACG 200
ATGTTCCTGT AATCGCTGGT TCAGCTTTAA GAGCTTTAGA AGGCGACGCT 250
KCATACGAAG AA.A.AAATTCTTGAATTGATG GCTGCAGTTG ACGAATATAT 300
CCCAACTCCA GAACGTGATA ACGACAAACC ATTCATGATG CCAGTTGAGG 350
ACGTATTCTC AATCACTGGT CGTGGTACTG TTGCTACAGG ACGTGTTGAA 400
CGTGGACAAG YTCGTGTTGG TGACGTTATC GATATCGTTG GTATCGCAGA 450
AGAAACAGCT CAAACAACTG TAACTGGTGT TGAAATGTTC CGTAAATTAT 500
TAGACTACGC TGAAGCAGGC GATAACATTG GTGCGTTACT ACGTGGTGTT 550
TCACGTGAAG ACATCCAACG TGGTCAAGTT TTAGCTAAAC CAGGTACAAT 600
CACACCTCAT ACAAAATTCT CTGCAGAAGT ATACGTGTTG ACTAAAGAAG 650
AAGGTGGACG TCATACTCCA TTCTTCACTA ACTACCGTCC ACAATTCTAC 700
TTCYGTACGA CTGACGTAAC TRGTGTTGTY GAATTACCAG AAGGAACTGA 750
AATGGTTATG CCTGGCGACA ACGTAACAAT GGAAGTTGAA TTAATCCACC 800
CAATCGCTAT CGAAAATGGT ACTAAATTCT CAATC 835
245
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID NO: 69
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 836 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus pseudoavium
(B) STRAIN: ATCC 49372
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 69
CGGAGCTATC TTAGTAGTAT CTGCTGCTGA TGGCCCTATG CCTCAAACAC 50
GTGAACACAT CTTGTTATCT CGTAACGTTG GTGTTCCTTA CATCGYTGTA 100
TTCTTAAACA AAATGGATAT GGTTGATGAC GAAGAATTAC TAGAATTAGT 150
TGAAATGGAA GTTCGTGACT TATTGTCAGA ATACGATTTC CCAGGCGACG 200
ACACTCYAGT TATCGCTGGT TCAGCYTTGA AAGCTTTAGA AGGCGACCCT 250
TCATACRAAG AAAAAATCTT AGAATTAATG SCTGCTGTTG ACGAATACAT 300
CCCAACACCA GTTCGTGATA CTGACAAACC ATTCATGATG CCAGTCGAAG 350
ACGTATTCTC AATCACTGGT CGTGGTACTG TTGCAACTGG TCGTGTTGAA 400
CGTGGACAAG TTCGCGTTGG TGACGAAGTT GAAATCGTAG GTATCGCTGA 450
AGAAACTGCT AAAACAACTG TTACAGGTGT TGAAATGTTC CGTAAATTGT 500
TAGACTACGC TGAAGCAGGC GATAACATCG GTGCATTATT ACGTGGTGTT 550
GCACGTGAAG ACATCCAACG TGGACAAGTA TTGGCTAAAC CAGCTTCAAT 600
CACTCCACAT ACAAAATTCT CTGCAGAAGT TTACGTTTTA ACTAA.AGAAG 650
AAGGCGGGCG TCACACTCCG TTCTTCACTA ACTACCGTCC TCAGTTCTAC 700
TTCCGTACAA CTGACGTAAC TGGTGTTGTT GATCTACCAG AAGGTACTGA 750
AATGGTAATG CCTGGTGATA ACGTAACTAT GGAAGTTGAA TTAATCCACC 800
CAATCGCGAT CGAAGACGGA ACTCGTTTCT CTATTC 836
2)INFORMATION FOR SEQ ID N0: 70
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus raffinosus
(B) STRAIN: ATCC 49427
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 70
CGGAGCTRTC TTAGTAGTAT CTGCTGCTGA TGGCCCTATG CCTCAAACTC 50
GTGAACACAT CTTGTTATCT CGTAACGTTG GTGTTCCTTA CATCGTTGTA 100
TTCTTAAACA AAATGGATAT GGTTGACGAT GAAGAATTAC TAGAATTAGT 150
TGAAATGGAA GTTCGTGACT TATTAACTGA ATACGACTTC CCAGGCGACG 200
ACACTCCAGT TATCGCAGGT TCAGCTTTGA AAGCCTTAGA AGGCGACGCT 250
TCATACGAAG F~AA.A.A.ATCTTAGAATTAATG GCTGCTGTTG ATGAATACAT 300
246
CA 02307010 2000-OS-19
CCCAACACCA GTTCGTGATA CTGACAAACC ATTCATGATG CCAGYGGAAG 350
ACGTAYTCTC AATCACTGGT CGTGGAACTG TTGCAACTGG TCGTGTTGAA 400
CGTGGACAAG TTCGCGTTGG TGACGAAGTT GAAATCGTAG GTATTGCTGA 450
AGAAACTGCT AAAACAACTG TTACAGGTGT TGAAATGTTC CGTAAATTGT 500
TGGATTACGC TGAAGCGGGC GACAACATTG GTGCATTATT ACGTGGTGTT 550
GCACGTGAAG ACATCCAACG TGGACAAGTA TTGGCTAAAC CAGCTTCAAT 600
CACTCCACAT ACAAAATTCT CTGCAGAAGT TTACGTTTTA ACTAAAGAAG 650
AAGGCGGACG TCATACTCCA TTCTTCACTA ACTACCGTCC TCAGTTCTAC 700
TTCCGTACAA CTGACGTAAC TGGTGTAGTT GATCTACCAG AAGGTACTGA 750
AATGGTAATG CCTGGTGATA ACGTAACTAT GGAAGTTGAA TTAATCCACC 800
CAATCGCGAT CGAAGACGGA ACTCGTTTCT CTATT 835
2)INFORMATION FOR SEQ ID N0: 71
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus saccharolyticus
(B) STRAIN: ATCC 43076
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 71
CGGCGCGATC TTAGTAGTAT CTGCTGCTGA TGGTCCTATG CCTCAAACTC 50
GTGAACACAT CTTGTTATCT CGTAACGTAG GTGTTCCTTA CATCGTTGTA 100
TTCTTAAACA AAATGGATAT GGTTGATGAC GAAGAATTAT TAGAATTAGT 150
AGAAATGGAA GTTCGTGACT TATTATCAGA ATACGATTTC CCAGGCGATG 200
ACACTCCAGT TATTGCAGGT TCTGCTTTGA AAGCTTTAGA AGGCGATCCA 250
GTTTACGAAG AAA.P~AATCTTCGAATTAATG GCTGCAGTTG ACGAATATAT 300
CCCAACTCCA GAACGTGATA CTGA.AA.AACCATTCATGATG CCAGTTGAGG 350
ATGTATTCTC AATCACTGGT CGTGGTACTG TTGCTACAGG TCGTGTTGAA 400
CGTGGACAAG TTCGCGTTGG TGACGTTGTA GAAATCGTTG GTATCGACGA 450
AGAAACAGCT CAAACTACTG TAACAGGTGT TGAAATGTTC CGTAAATTAT 500
TAGACTACGC TGAAGCAGGC GATAACATCG GTGCTTTATT ACGTGGGGTT 550
GCTCGTGAAG ACATCCAACG TGGACAAGTA TTAGCTAAAC CAGGAACAAT 600
CACTCCTCAT ACAAAATTCG TAGCTGAAGT TTACGTTTTA ACTAAAGAAG 650
AAGGTGGACG TCATACTCCA TTCTTCACTA ACTACCGTCC TCAATTCTAC 700
TTCCGTACAA CTGACGTAAC TGGTGTTGTA GAATTACGCG AAGGTACTGA 750
AATGGTAATG CCTGGTGACA ACGTAACTAT CGACGTTGAA TTAATCCACC 800
CAATCGCTAT CGAAGACGGA ACTCGTTTCT CTATT 835
2)INFORMATION FOR SEQ ID N0: 72
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 823 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
247
CA 02307010 2000-OS-19
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus solitarius
(B) STRAIN: ATCC 49428
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 72
GAGCTATCTT GGTAGTTTCT GCAGCTGATG GCCCAATGCC ACAAACTCGT 50
GAACATATTT TGTTGTCACG TAATGTAGGT GTACCTTACA TCGTTGTGTT 100
CTTGAACAAA ATGGATATGG TTGATGACGA AGAATTACTT GAGTTAGTTG 150
AAATGGAAGT ACGTGATCTA TTATCTGAAT ACGACTTCCC AGGAGATGAT 200
ACTCCAGTTA TTTCCGGTTC AGCTTTGAAA GCTTTAGAAG GCGACGAAGA 250
ATATGAACAA AAAATTATGG ACTTAATGGA TGCAGTTGAT GACTACATTC 300
CAACTCCTGA ACGTGACCAT GACAAACCAT TCATGATGCC AATTGAAGAT 350
GTATTTTCAA TTACAGGCCG TGGTACTGTT GCTACAGGAC GTGTTGAACG 400
CGGGACTATC AAAGTCGGCG ATGAAGTTGA CATTATTGGT ATTCATGAAG 450
ACGTTAAA.AA GACAACAGTT ACTGGTGTAG AAATGTTCCG TAAATTGTTG 500
GACTACGCTG AAGCAGGCGA TAACATTGGT ACTTTGTTAC GTGGTGTTTC 550
TCGTGATGAT ATCGAACGTG GTCAAGTATT AGCTAAACCA GGTTCAATCA 600
CACCACATAC AAGATTCTCT GCTGAAGTTT ATGTTTTGAC TAAAGAAGAA 650
GGCGGACGTC ATACTCCATT CTTCTCAAAC TATCGTCCTC AATTCTACTT 700
CCGTACAACT GATATCACTG GTGTCATTGA ATTGCCAGAA GGTACTGAAA 750
TGGTAATGCC AGGTGATAAT GTAACAATGG ATGTTGAATT AATCCACCCA 800
GTCGCTATCG AAGAAGGAAC TCG 823
2)INFORMATION FOR SEQ ID N0: 73
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus casseliflavus
(B) STRAIN: ATCC 25788
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 73
CGGTGCAATC TTGGTCGTAT CAGCGACAGA TGGCCCAATG CCACAAACAC 50
GGGAGCATAT TTTGCTTTCT CGTCAAGTGG GTGTGAAACA TTTGATCGTC 100
TTTTTGAATA AGACGGACCT TGTCGATGAT GACGAGTTGA TCGATTTAGT 150
TGAAATGGAA GTCAGAGAAT TGCTGACTGA ATATGATTTT CCTGGCGACG 200
ACATTCCTGT GATCAAGGGC TCTGCGTTAA AAGCCTTGGA AGGGGACCCA 250
GATGCTGAAG CAGCGATCTT AACGCTGATG GATACAGTAG ATGAATATAT 300
CCCAACGCCA GAACGTGATA CTGACAAACC ATTGTTGTTA CCGATCGAAG 350
ATGTCTTTTC GATCACAGGA CGGGGGACCG TTGCTTCTGG TCGGATCGAT 400
CGCGGCATGG TAAAAGTCGG GGATGAAGTA GAAATCGTCG GAATCAAACC 450
TGAAACACAA AAAGCAGTCG TGACAGGGGT AGAAATGTTC CGCAAAACGA 500
TGGACTTCGG AGAAGCTGGC GATAACGTAG GGGTATTGTT ACGGGGCATC 550
ACCCGTGATG AAATTGAACG TGGCCAAGTG TTAGCAAAAC CAGGTTCTAT 600
CACACCGCAT ACGAAATTCC AAGCGGAAGT CTATGTGTTG ACAAAAGAAG 650
AAGGCGGTCG CCATACCCCA TTCTTTAATA ATTATCGCCC ACAATTTTAC 700
TTCCGTACAA CGGACGTAAC TGGGAATATC GTTTTACCAG AAGGAACGGA 750
AATGGTGATG CCTGGTGACA ACGTAACGAT CGATGTGGAA TTGATCCATC 800
CGATCGCTGT AGAAAATGGA ACGACCTTCT CGATT 835
248
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID N0: 74
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 380 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Staphylococcus saprophyticus
(B) STRAIN: ATCC 15305
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 74
TAACGGGCGT CTCGATAGAA AAACACGTGA AAATCCCAAT GATTATAAAC 50
AATCAATATA CGATTTTGCT GAAGCTGTAA CAAAAGGTAT TAAGGAACAA 100
ACAAATAAAA ATTAATAGGC AACTTAACCA GAATCGTTAA AACTATATGA 150
AGATTCTGGT TTTTTAAATT CAAAAAGTTT TCTAAP~AAATTTACTTGCTT 200
TTTTAAGTAT AGGTATAAAA TACGATTGAT TAAAACAGTA AAGGAAATGA 250
ATCATGAAAC AATTAACTAA GCCTTTATAC TTTTACCTAT TACTTTTTAT 300
TACAACAACG CTGATTGGCG CGTTACTATT ATATTTGCCA ATCACAGGTA 350
AACATCCTAT TGATTTTGTG GACGCCCGTT 380
2)INFORMATION FOR SEQ ID N0: 75
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 666 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus flavescens
(B) STRAIN: ATCC 49996
(xi)SEQUENCE
DESCRIPTION:
SEQ ID NO:
75
GCAATCTTGG TCGTATCAGC GACAGATGGC CCAATGCCAC AAACACGGGA 50
GCATATTTTG CTTTCTCGTC AAGTGGGTGT GAAACATTTG ATCGTCTTTT 100
TGAATAAGAC GGACCTTGTC GATGATGACG AGTTGATCGA TTTAGTTGAA 150
ATGGAAGTCA GAGAATTGCT GACTGAATAT GATTTTCCTG GCGACGACAT 200
TCCTGTGATC AAGGGCTCTG CGTTAAAAGC CTTGGAAGGG GACCCAGATG 250
CTGAAGCAGC GATCTTAACG CTGATGGATA CGGTAGATGA ATATATCCCA 300
ACGCCAGAAC GTGATACTGA CAAACCATTG TTGTTACCGA TCGAAGATGT 350
CTTTTCGATC ACAGGACGGG GGACCGTTGC TTCTGGTCGG ATCGATCGCG 400
GCATGGTAAA AGTCGGGGAT GAAGTAGAAA TCGTCGGAAT CAAACCTGAA 450
ACACAP~AAAG CAGTCGTGAC AGGGGTAGAA ATGTTCCGCA AAACGATGGA 500
CTTCGGAGAA GCTGGCGATA ACGTAGGGGT ATTGTTACGG GGCATCACCC 550
GTGATGAAAT TGAACGTGGC CAAGTGTTAG CAAAACCAGG TTCTATCACA 600
CCGCATACGA AATTCCAAGC GGAAGTCTAT GTGTTGACAA AAGAAGAAGG 650
CGGTCGCCAT ACCCCA 666
249
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID NO: 76
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 751 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Enterococcus gallinarum
(B) STRAIN: ATCC 49573
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 76
TGGTGCGATT TTAGTTGTAT CCGCAACAGA TGGTCCAATG CCTCAAACCC 50
GGGAACATAT CTTGCTTTCG AGACAAGTTG GTGTGAAACA TCTGATTGTT 100
TTCTTGAACA AAATCGATTT AGTCGATGAC GAAGAATTGA TTGATTTAGT 150
AGAAATGGAA GTAAGAGAAC TGCTATCTGA ATATAATTTT CCAGGGGATG 200
ACATTCCTGT TATCAAAGGT TCGGCATTAA AAGCGTTGGA AGGAGACCCT 250
GATGCAGAAG CTGCCATCAT GGAATTAATG GATACAGTAG ACAGCTATAT 300
CCCAACACCT GAGCGTGATA CAGACAAACC ATTACTCTTG CCAGTTGAAG 350
ATGTCTTTTC GATTACTGGA CGAGGAACAG TTGCTTCCGG ACGGATCGAT 400
CGGGGAACAG TTCGGGTAGG CGATGAAGTA GAAATCGTCG GTATCAAACC 450
TGAAACCCAA AAAGCTGTAG TGACAGGCGT CGAA.ATGTTCCGCAAGACGA 500
TGGACTTTGG GGAAGCCGGT GACAATGTAG GTGTCTTGCT GAGAGGGATC 550
ACTCGTGACG AAATTGAACG AGGACAAGTG TTGGCTAAAC CAGGTTCGAT 600
CACACCACAT ACAAAATTCC AAGCAGAAGT TTATGTATTG ACGAAAGAAG 650
AAGGTGGTCG TCATACACCA TTCTTCAACA ACTATCGTCC ACAATTTTAT 700
TTCCGTACAA CGGATGTGAC AGGGAACATT ACATTGCCTG AAGGAACAGA 750
A 751
2)INFORMATION FOR SEQ ID NO: 77
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 834 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Ehrlichia cam s
(B) STRAIN: Florida
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 77
TGCAGCAATA TTAGTAGTGT CTGCAACTGA TGGAGCAATG CCACAAACAA 50
GAGAACATAT ATTATTAGCA AAGCAAGTAG GTGTAAAAGA TATAGTAGTG 100
TGGATGAATA AGTGTGATGT TGTAGATGAT GAAGAAATGT TGTCATTAGT 150
TGAAATGGAA ATAAGGGAAT TGTTATCAAA ATATGGGTAT CCTGGGGATG 200
ATATAGATGT AGTTAGAGGA TCTGCAGTTA AAGCATTAGA AGAAGAAACA 250
GGCTCAGGTG TGTGGAGTGA AAAAATAATG GAATTGATGA ATGCTTTAGA 300
AAAAATAAGT TTACCAGTAA GAGAAAAAGA TAAGCCATTT TTAATGTCAA 350
250
CA 02307010 2000-OS-19
TAGAAGATGT GTTTTCAATA CCTGGAAGAG GTACAGTAGT AACAGGAAGA 400
ATAGAAAGAG GAGTAATTAG AGTAGGGGAT AAAATAGAGA TAGTAGGATT 450
GCGTGAGATA CAAAGTACAG TATGTACAGG TGTTGAAATG TTTCATAAAG 500
CATTAGATGC AGGAGAAGCA GGGGATAATG CTGGAATATT GTTAAGAGGG 550
ATAAA.AAAAG AAGATGTAGA AAGAGGGCAA GTATTGAGTG CACCTGGACA 600
GATACATTCA TATAAGAGAT TTAAGGCAGA GGTATATATA TTGAAAAAAG 650
AAGAAGGAGG AAGACATACT CCATTTTTCT CAAATTACCA GCCGCAATTT 700
TATGTTAGAA CAACAGATGT AACAGGGAAT ATAAAGTTAC CAGAAGGAGT 750
AGAAATGGTA ATGCCAGGGG ATAATATAAA TATCGAAGTG AGTTTGGATA 800
AGCCTGTTGC TATTGATCAA GGATTGAGAT TTGC 834
2)INFORMATION FOR SEQ ID N0: 78
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 817 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli
(B) STRAIN: ATCC 23511
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 78
CGGCGCGATC CTGGTAGTTG CTGCGACTGA CGGCCCGATG CCGCAGACTC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCGTA CATCATCGTG 100
TTCCTGAACA AATGCGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
TGAAATGGAA GTTCGTGAAC TTCTGTCTCA GTACGACTTC CCGGGCGACG 200
ACACTCCGAT CGTTCGTGGT TCTGCTCTGA AAGCGCTGGA AGGCGACGCA 250
GAGTGGGAAG CGAAAATCCT GGAACTGGCT GGCTTCCTGG ATTCTTAYAT 300
TCCGGAACCA GAGCGTGCGA TTGACAAGCC GTTCCTGCTG CCGATCGAAG 350
ACGTATTCTC CATCTCCGGT CGTGGTACCG TTGTTACCGG TCGTGTAGAA 400
CGCGGTATCA TCAAAGTTGG TGAAGAAGTT GAAATCGTTG GTATCAAAGA 450
GACTCAGAAG TCTACCTGTA CTGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGCCG TGCTGGTGAG AACGTAGGTG TTCTGCTGCG TGGTATCAAA 550
CGTGAAGAAA TCGAACGTGG TCAGGTACTG GCTAAGCCGG GCACCATCAA 600
GCCGCACACC AAGTTCGAAT CTGAAGTGTA CATTCTGTCC AAAGATGAAG 650
GCGGCCGTCA TACTCCGTTC TTCAAAGGCT ACCGTCCGCA GTTCTACTTC 700
CGTACTACTG ACGTGACTGG TACCATCGAA CTGCCGGAAG GCGTAGAGAT 750
GGTAATGCCG GGCGACAACA TCAAAATGGT TGTTACCCTG ATCCACCCGA 800
TCGCGATGGA CGACGGT 817
2)INFORMATION FOR SEQ ID NO: 79
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 825 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
251
CA 02307010 2000-OS-19
(A) ORGANISM: Escherichia fergusonii
(B) STRAIN: ATCC 35469
(xi)SEQUENCE
DESCRIPTION:
SEQ ID N0:
79
CGATCCTGGT AGTTGCTGCG ACTGACGGCC CGATGCCGCA GACTCGTGAG 50
CACATCCTGC TGGGTCGTCA GGTAGGCGTT CCGTACATCA TCGTGTTCCT 100
GAACAAGTGC GACATGGTTG ATGACGAAGA GCTGCTGGAA CTGGTTGAAA 150
TGGAAGTTCG TGAACTTCTG TCTCAGTACG ACTTCCCGGG CGACGACACT 200
CCGATCGTTC GTGGTTCTGC TCTGAAAGCG CTGGAAGGCG ACGCAGAGTG 250
GGAAGCGAAA ATCCTGGAAC TGGCTGGCTT CCTGGATTCT TACATTCCGG 300
AACCAGAGCG TGCGATTGAC AAGCCGTTCC TGCTGCCGAT CGAAGACGTG 350
TTCTCCATCT CCGGTCGTGG TACCGTTGTT ACCGGTCGTG TAGAACGCGG 400
TATCATCAAA GTTGGTGAAG AAGTTGAAAT CGTTGGTATC AAAGAGACTC 450
AGAAGTCTAC CTGTACTGGC GTTGAAATGT TCCGCAAACT GCTGGACGAA 500
GGCCGTGCTG GTGAGAACGT AGGTGTTCTG CTGCGTGGTA TCAAACGTGA 550
AGAAATCGAA CGTGGTCAGG TACTGGCTAA GCCGGGCACC ATCAAGCCGC 600
ACACCAAGTT CGAATCTGAA GTGTACATTC TGTCCAAAGA TGAAGGCGGT 650
CGTCATACTC CGTTCTTCAA AGGCTACCGT CCGCAGTTCT ACTTCCGTAC 700
TACTGACGTG ACTGGTACCA TCGAACTGCC GGAAGGCGTA GAGATGGTAA 750
TGCCGGGCGA CAACATCAAA ATGGTTGTTA CCCTGATCCA CCCGATCGCG 800
ATGGACGACG GTCTGCGTTT CGCAA 825
2)INFORMATION FOR SEQ ID NO: 80
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 829 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Escherichia hermannii
(B) STRAIN: ATCC 33650
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 80
GGCGCGATCC TGGTTGTTGC TGCGACTGAC GGCCCGATGC CGCAGACCCG 50
TGAGCACATC CTGCTGGGTC GTCAGGTAGG CGTTCCGTAC ATCATCGTGT 100
TCCTGAACAA ATGCGACATG GTTGATGACG AAGAGCTGCT GGAACTGGTT 150
GAGATGGAAG TTCGCGAACT GCTGTCCCAG TACGATTTCC CGGGCGACGA 200
CACCCCGATC GTTCGTGGTT CCGCGCTGAA AGCGCTGGAA GGCGAAGCAG 250
AGTGGGAAGA GAAAATCATC GAACTGGCTG GCTACCTGGA TTCCTATATC 300
CCGGAACCAG AGCGTGCGAT TGACAAGCCG TTCCTGCTGC CTATCGAAGA 350
CGTATTCTCC ATCTCCGGCC GTGGTACCGT TGTTACCGGT CGTGTAGAGC 400
GCGGTATCAT CAAAGTGGGT GAAGAAGTTG AAATCGTGGG TATCAAAGAT 450
ACTGCGAAAT CAACCTGTAC CGGCGTTGAA ATGTTCCGCA AACTGCTGGA 500
CGAAGGCCGT GCGGGCGAGA ACGTGGGTGT TCTGCTGCGT GGTATCAAAC 550
GTGAAGAAAT CGAACGTGGT CAGGTACTGG CTAAGCCGGG TTCCATCAAG 600
CCKCACACCA AGTTCGAATC TGAAGTGTAC ATTCTGTCCA AAGACGAAGG 650
CGGCCGTCAC ACTCCGTTCT TCAAAGGCTA CCGTCCGCAG TTCTACTTCC 700
GTACAACTGA CGTGACTGGC ACCATCGAAC TGCCGGAAGG CGTTGAGATG 750
GTAATGCCGG GCGACAACAT CAAAATGGTT GTTACCCTGA TCCACCCGAT 800
CGCGATGGAC GACGGTCTGC GTTTCGCAA 829
252
CA 02307010 2000-OS-19
2)INFORMATION FOR SEQ ID N0: 81
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 816 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Escherichia vulneris
(B) STRAIN: ATCC 33821
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 81
CGGCGCGATC CTGGTTGTTG CTGCGACTGA CGGCCCGATG CCGCAGACCC 50
GTGAGCACAT CCTGCTGGGT CGTCAGGTAG GCGTTCCGTA CATCATCGTG 100
TTCCTGAACA AATGCGACAT GGTTGATGAC GAAGAGCTGC TGGAACTGGT 150
TGAGATGGAA GTGCGTGAAC TTCTGTCCCA GTACGACTTC CCGGGCGACG 200
ACACCCCGAT CATTCGTGGT TCTGCGCTGA AAGCGCTGGA AGGCGAAGCT 250
GAGTGGGAAG AGAAAATCGT TGAGCTGGCT GGCTACCTGG ATTCCTACAT 300
CCCGGAACCA GAGCGTGCGA TTGACAAGCC GTTCCTGCTG CCGATCGAAG 350
ACGTATTCTC CATCTCCGGT CGTGGTACCG TTGTTACCGG TCGTGTAGAG 400
CGCGGTATCA TCAARGTKGG TGAAGAAGTT GAAATCGTGG GTATCAAAGA 450
TACTGCGAAA TCTACCTGTA CCGGCGTTGA AATGTTCCGC AAACTGCTGG 500
ACGAAGGTCG TGCAGGCGAG AACTGCGGCG TTCTGCTGCG TGGTATCAAG 550
CGTGAAGAGA TCCAGCGTGG CCAGGTTCTG GCTAAGCCGG GCTCAATCAA 600
GCCGCACACC AAGTTCGAAT CCGAAGTGTA CATCCTGTCC AAAGACGAAG 650
GCGGCCGTCA CACTCCGTTC TTCAAAGGCT ACCGTCCGCA GTTCTACTTC 700
CGTACAACTG ACGTGACTGG CACCATCGAA CTGCCGGAAG GCGTAGAGAT 750
GGTAATGCCG GGCGACAACA TCAAAATGGT TGTTACCCTG ATCCATCCGA 800
TCGCGATGGA CGACGG 816
2)INFORMATION FOR SEQ ID NO: 82
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 828 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Eubacterium lentum
(B) STRAIN: ATCC 43055
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 82
CGGCGCCTCC TCGTTATCGC CGCCACCGAC GGCCCGATGG CCCAGACCCG 50
CGAGCACATC CTGCTCGCCC GTCAGGTCGG CGTGCCCTAC ATCGTGGTCT 100
TCCTGAACAA GTGCGACATG GTCGACGACG AGGAGCTCCT CGAGCTCGTC 150
GAGATGGAAG TTCGCGAGCT GCTCGACTCT TACGAGTTCC CGGGCGACGA 200
CACCCCGATC ATCCGCGGCT CCGCTTTGAA GGCCCTCGAG GGCGACAAAG 250
AGTGGCAGGA GAAGGTCTGG GAGCTCATGG ACGCCGTCGA CTCCTACATC 300
CCGACGCCGG AGCGCATGGT CGACAAGCCG TTCCTGATGG CCGTCGAGGA 350
253
CA 02307010 2000-OS-19
CACGATGACC ATCACCGGCC GCGGCACCGT TGCCACCGGT CGTGTGGAGC 400
GTGGTACGCT GCATGTCAAC GACCCGCTGG AGATCGTCGG TATCAAGGAG 450
ACCCAGAACA CGGTCTGCAC CGGTATCGAG ATGTTCCGCA AGCTGCTCGA 500
CGAGGCTCAG GCCGGCGACA ACATCGGCTG CCTGCTCCGC GGTGTCAAGC 550
GCGAGGAGAT CGTTCGCGGC CAGGTTCTCT GCAAGCCCGG TAGCGTGACC 600
CCGCACACCG AGTTCGAGGG TCAGGTCTAC ATCCTGACGA AGGAAGAGGG 650
CGGCCGCCAC ACGCCGTTCT TCGACGGCTA CCGTCCGCAG TTCTACTTCC 700
GCACGACGGA CGTGACGGGT GTTGCCCACC TTCCCGAGGG CACCGAGATG 750
GTCATGCCGG GCGACAACGT GGAGATCAAG GGCGAGCTCA TTCACCCGAT 800
CGCCAGGAAG AGGGCTGCGT TCGCTAAC 828
2)INFORMATION FOR SEQ ID N0: 83
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 835 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Eubacterium nodatum
(B) STRAIN: ATCC 33099
(xi)SEQUENCE DESCRIPTION: SEQ ID N0: 83
GGAGCAATTC TGGTTTGTGC AGCAACKGAC GGACCAATGC CTCAGACAAG 50
AGAACATATC CTTTTGTCAA GGCAGGTAGG AGTGCCATAT ATCATCGTAT 100
TCCTGAATAA ATGTGACATG GTGGATGAYG AAGAGCTTCT GGACTTGGTA 150
GAGATGGAAG TAAGAGAACT TCTCAGTGAG TATGAATTCC CGGGAGATGA 200
TACCCCGATA GTAAGAGGTT CAGCCCTGAA GGCACTGGAA GAACCCAATG 250
GAGAATGGGC AGACAAGATT GTAGAGCTGA TGGAGGAAGT AGATAAATAC 300
ATTCCTGAAC CAAAGAGAGA TAACGACAAA CCGTTCCTGA TGCCTGTAGA 350
GGACGTATTC TCAATAACAG GAAGAGGAAC AGTAGCGACA GGAAGRGTTG 400
AAAGAGGAAT CCTGAAGGTC GGTGATGAAG TAGAAATCGT GGGAATGAGC 450
GAAGAGAGAA GAAAGGTAGT AGTAACGGGA GTTGAAATGT TCAGAAAGCT 500
TCTGGATGAA GCAGAGACAG GAGACAACAT CGGAGCACTG CTGAGAGGAG 550
TTCAGAGAAC RGAGATCCAG AGAGGTCAGG TATTGGCRGC ACCTGGAACG 600
ATCAACCCAC ATACAAAGTT CAAGGGTCAG GTATATGTAC TGAAGAAGGA 650
AGAAGGAGGA AGGCATACGC CGTTCTTCAA YGGATACAGW CCACAGTTCT 700
ACTTCAGAAC AACAGACGTA ACAGGAGATT TGCAGCTGCC GGAAGGARCA 750
GAGATGTGCA TGCCGGGAGA TAATGTGGTA ATGAACRTCA GCCTGATCAC 800
TCCGATTGCT ATAGAAGAGG GWCTGAGATT TGCCA 835
2)INFORMATION FOR SEQ ID N0: 84
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 bases
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(ii)MOLECULE TYPE: Genomic DNA
(vi)ORIGINAL SOURCE:
254
CA 02307010 2000-OS-19' .
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PART1E DE CETTE DEMANDE OU CE BREVET
COMPREND PLUS D'UN TOME.
CECI EST LE TOME ~ DE ~_
NOTE: Pour les tomes additioneis, veuiliez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICAT10NlPATENT CONTAINS MORE
THAN ONE VOLUME
. THIS IS VOLUME y~__ OF
(.
' NOTE: For additional volumes-phase contact the Canadian Patent Office .
