Note: Descriptions are shown in the official language in which they were submitted.
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Modified microbial nucleic acid for use in detection and analysis of
microorganisms
Technical Field
The invention relates to modified nucleic acid for use in detection and
analysis of
microorganisms.
Background Art
A number of procedures are presently avaiiable for the detection of specific
nucleic acid molecules. These procedures typically'depend on sequence-
dependent
hybridisation between the target nucleic acid and nucleic acid probes which
may range in
length from short oligonucleotides (20 bases or less) to sequences of many
kilobases (kb).
The most widely used method for amplification of specific sequences from
within
a population of nucleic acid sequences is that of polymerase chain reaction
(PCR)
(Dieffenbach, C and Dveksler, G. eds. PCR Primer: A Laboratory Manual. Cold
Spring
Harbor Press, Plainview NY). In this amplification method, oligonucleotides,
generally 20
to 30 nucleotides in length on complementary DNA strands and at either end of
the
region to be amplified, are used to prime DNA synthesis on denatured single-
stranded
DNA. Successive cycles of denaturation, primer hybridisation and DNA strand
synthesis
using thermostable DNA polymerases allows exponential amplification of the
sequences
between the primers. RNA sequences can be amplified by first copying using
reverse
transcriptase to produce a complementary DNA (cDNA) copy. Amplified DNA
fragments
can be detected by a variety of means including gel electrophoresis,
hybridisation with
labelled probes, use of tagged primers that allow subsequent identification
(eg by'an
enzyme linked assay), and use of fluorescently-tagged primers that give rise
to a signal
upon hybridisation with the target DNA (eg Beacon and TaqMan systems).
As well as PCR, a variety of other techniques have been developed for
detection
and amplification of specific nucleotide sequences. One example is the ligase
chain
reaction (1991, Barany, F. et af., Proc. Nati. Acad. Sci. USA 88, 189-193).
Another example is isothermal amplification which was first described in 1992
(Walker GT, Little MC, Nadeau JG and Shank D. Isothermal in vitro
amplification of DNA
by a restriction enzyme/DNA polymerase system. PNAS 89: 392-396 (1992) and
termed
Strand Displacement Amplification (SDA). Since then, a number of other
isothermal
amplification technologies have been described including Transcription
Mediated
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Amplification (TMA) and Nucleic Acid Sequence Based Amplification (NASBA) that
use
an RNA polymerase to copy RNA sequences but not corresponding genomic DNA'
(Guatelli JC, Whitfield KM, Kwoh DY, Barringer KJ, Richmann DD and Gingeras
TR.
Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction
modeled after
retroviral replication. PNAS 87: 1874-1878 (1990): Kievits T, van Gemen B, van
Strijp D,
Schukkink R, Dircks N1, Adriaanse H, Malek L, Sooknanan R, Lens P. NASBA
isothermal
enzymatic in vitro nucleic acid amplification optimized for the diagnosis of
HIV-1
infection. J Virol Methods. 1991 Dec; 35(3):273-86).
Other DNA-based isothermal techniques include Rolling Circle Amplification
(RCA)
in which a DNA polymerase 'extends a primer directed to a circular template
(Fire A and
Xu SQ. Rolling replication of short circles. PNAS 92: 4641-4645 (1995),
Ramification
Amplification (RAM) that uses a circular probe for target detection (Zhang W,
Cohenford
M, Lentrichia B, Isenberg HD, Simson E, Li H, Yi J, Zhang DY. Detection of
Chlamydia
trachomatis by isothermal ramification amplification method: a feasibility
study. J Clin
Microbiol. 2002 Jan; 40(1):128-32.) and more recently, Helicase-Dependent
isothermal
DNA amplification (HDA), that uses a helicase enzyme to unwind the DNA strands
instead of heat (Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA
amplification. EMBO Rep. 2004 Aug; 5(8):795-800.)
Recently, isothermal methods of DNA amplification have been described (Walker
GT, Little MC, Nadeau JG and Shank D. Isothermal in vitro amplification of DNA
by a
restriction enzyme/DNA polymerase system. PNAS 89: 392-396 (1992). Traditional
amplification techniques rely on continuing cycles of denaturation and
renaturation of the
target molecules at each cycle of the amplification reaction. Heat treatment
of DNA
results in a certain degree of shearing of DNA molecules, thus when DNA is
limiting such
as in the isolation of DNA from a small number of cells from a developing
blastocyst, or
particularly in cases when the DNA is already in a fragmented form, such as in
tissue
sections, paraffin blocks and ancient DNA samples, this heating-cooling cycle
could
further damage the DNA and result in loss of amplification signals. Isothermal
methods
do not rely on the continuing denaturation of the template DNA to produce
single
stranded molecules to serve as templates from further amplification, but on
enzymatic
nicking of DNA molecules by specific restriction endonucleases at a constant
temperature:
The technique termed Strand Displacement Amplification (SDA) relies on the
ability of certain restriction enzymes to nick the unmodified strand of hemi-
modified DNA
and the ability of a 5'-3' exonuclease-deficient polymerase to extend and
displace the
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downstream strand. Exponential amplification is then achieved by coupling
sense and
antisense reactions in which strand displacement from the sense reaction
serves as a
template for the antisense reaction (Walker GT, Little MC, Nadeau JG and Shank
D.
Isothermal in vitro amplification of DNA by a restriction enzyme/DNA
polymerase system.
PNAS 89: 392-396 (1992). Such techniques have been used for the successful
amplification of Mycobacterium tuberculosis (Walker GT, Little MC, Nadeau JG
and
Shank D. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA
polymerase system. PNAS 89: 392-396 (1992), HIV-1, Hepatitis C and HPV-16
Nuovo
G. J., 2000), Chlamydia trachomatis (Spears PA, Linn P, Woodard DL and Walker
GT.
Simultaneous Strand Displacement Amplification and Fluorescence Polarization
Detection of Chlamydia trachomatis. Anal. Biochem. 247: 130-137 (1997).
The use of SDA to date has depended on modified phosphorthioate nucleotides in
order to produce,a hemi-phosphorthioate DNA duplex that on the modified strand
would
be resistant to enzyme cleavage, resulting in enzymic nicking instead of
digestion to
drive the displacement reaction. Recently, however, several "nickase" enzyme
have
been engineered. These enzymes do not cut DNA in the traditional manner but
produce
a nick on one of the DNA strands. "Nickase" enzymes include N.Alwl (Xu Y,
Lunnen KD
and Kong H. Engineering a nicking endonuclease N.Alwl by domain swapping. PNAS
98: 12990-12995 (2001), N.BstNB1 (Morgan RD, Calvet C, Demeter M, Agra R, Kong
H.
Characterization of the specific DNA nicking activity of restriction
endonuclease
N.BstNBI. Biol Chem. 2000 Nov;381(11):1123-5.) and M(y1 (Besnier CE, Kong H.
Converting M!yl endonuclease into a nicking enzyme by changing its
oligomerization
state. EMBO Rep. 2001 Sep;2(9):782-6. Epub 2001 Aug 23). The use of such
enzymes
would thus simplify the SDA procedure.
In addition, SDA has been improved by the use of a combination of a heat
stable
restriction enzyme (Aval) and Heat stable Exo-polymerase (Bst polymerase).
This
combination has been shown to increase amplification efficiency of the
reaction from a
108 fold amplification to 1010 foid amplification so that it is possible,
using this technique,
to the amplification of unique single copy molecules. The resultant
amplification factor
using the heat stable polymerase/enzyme combination is in the order of 109
(Milla M. A.,
Spears P. A., Pearson R. E: and Walker G. T. Use of the Restriction Enzyme
Aval and
Exo-Bst Polymerase in Strand Displacement Amplification Biotechniques 1997
24:392-
396).
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To date, all isothermal DNA amplification techniques require the initial
double
stranded template DNA molecule to be denatured prior to the initiation of
amplification.
In addition, amplification is only initiated once from each priming event.
For direct detection, the target nucleic acid is most commonly separated on
the
basis of size by gel electrophoresis and transferred to a solid support prior
to
hybridisation with a probe complementary to the target sequence (Southern and
Northern blotting). The probe may be a natural nucleic acid or analogue such
as peptide
nucleic acid (PNA) or locked nucleic acid (LNA) or intercalating nucleic acid
(INA). The
probe may be directly labelled (eg with 32P) or an indirect detection
procedure may be
used. Indirect procedures usually rely on incorporation into the probe of a
"tag" such as
biotin or digoxigenin and the probe is then detected by means such as enzyme-
linked
substrate conversion or chemiluminescence.
Another method for direct detection of nucleic acid that has been used widely
is
"sandwich" hybridisation. In this method, a capture probe is coupled to a
solid support
and the target nucleic acid, in solution, is hybridised with the bound probe.
Unbound
target nucleic acid is washed away and the bound nucleic acid is detected
using a
second probe that hybridises to the target sequences. Detection may use direct
or
indirect methods as outlined above. Examples of such methods include the
"branched
DNA" signal detection system, an example that uses the sandwich hybridization
principle
(1991, Urdea, M. S., et al., Nucleic Acids Symp. Ser. 24,197-200). A rapidly
growing
area that uses nucleic acid hybridisation for direct detection of nucleic acid
sequences is
that of DNA microarrays, (2002, Nature Genetics, 32, [Supplement]; 2004, Cope,
L.M., et
al., Bioinformatics, 20, 323-331; 2004, Kendall, S.L., et al., Trends in
Microbiology, 12,
537-544). In this process, individual nucleic acid species, that may range
from short
oligonucleotides, (typically 25-mers in the Affymetrix system), to longer
oligonucleotides,
(typically 60-mers in the Applied Biosystems and Agilent platforms), to even
longer
sequences such as cDNA clones, are fixed to a solid support in a grid pattern
or
photolithographically synthesized on a solid support. A tagged or labelled
nucleic acid
population is then hybridised with the array and the level of hybridisation to
each spot in
the array quantified. Most commonly, radioactively- or fluorescently-labelled
nucleic
acids (eg cRNAs or cDNAs) are used for hybridisation, though other detection
systems
can be employed, such as chemiluminescence.
A rapidly growing area that uses nucleic acid hybridisation for direct
detection of
nucleic acid sequences is that of DNA micro-arrays (Young RA Biomedical
discovery
with DNA arrays. Cell 102: 9-15 (2000); Watson A New tools. A new breed of
high tech
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detectives. Science 289:850-854 (2000)). In this process, individual nucleic
acid
species, that may range from oligonucleotides to longer sequences such as
complementary DNA (cDNA) clones, are fixed to a solid support in a grid
pattern. A
tagged or labelled nucleic acid population is then hybridised with the array
and the level
5 of hybridisation with each spot in the array quantified. Most commonly,
radioactively- or
fluorescently-labelled nucleic acids (eg cDNAs) were used for hybridisation,
though other
detection systems were employed.
Traditional methods for the detection of microorganisms such as bacteria,
yeasts
and fungi and include culture of the microorganisms on selective nutrient
media then
classification of the microorganism based on size, shape, spore production,
characters
such as biochemical or enzymatic reactions and specific staining properties
(such as the
Gram stain) as seen under conventional light microscopy. Viral species have to
be
grown in specialised tissue or cells then classified based on their structure
and size
determined by electron microscopy. A major drawback of such techniques is that
not all
microorganisms will grow under conventional culture or cell conditions
limiting the
usefulness of such approaches. With bacteria, for example, such as Neisseria
meningitidis, Streptococcus pneumoniae and Haemophilus influenzae (which all
cause
meningitis and amongst which N. meningitidis causes both meningitis'and
fulminant
meningococcaemia) all three species are difficult to culture. Blood culture
bottles are
routinely examined every day for up to seven days, and subculturing is
required.
H. influenzae requires special medium containing both nicotinamide adenine
dinucleotide
and haemin and growth on Chocolate Agar Plates. Blood cultures require
trypticase soy
broth or brain heart infusion and the addition of various additives such as
sodium
polyanetholesulphonate. For microorganisms such as Clostridium botulinum,
which
causes severe food poisoning and floppy baby syndrome, the identification of
the toxin
involves injection of food extracts or culture supernatants into mice and
visualization of
results after 2 days. In addition, culturing of the potential microorganism on
special
media takes a week. Staphylococcus aureus enterotoxin (a cause of food
poisoning as
well as skin infections, b(ood infections, pneumonia, osteomyelitis, arthritis
and bra.in
abscesses) is detected in minute amounts by selective absorption of the toxin
via ion
exchange resins or Reverse Passive Latex Agglutination using monoclonal
antibodies.
Its relative, S. epidermis, leads to blood infections and contaminates
equipment and
surfaces in hospitals and health care machines and appliances.
Non-viral, microorganisms can also be classified based on their metabolic
properties such as the production of specific amino acids or metabolites
during
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fermentation reactions on substrates such as glucose, maltose or sucrose.
Alternatively,
microorganisms can be typed based on their sensitivity to antibiotics.
Specific antibodies
to cell surface antigens or excreted proteins such as toxins are also used to
identify or
type microorganisms. However, all the above methods rely on the culture of the
microorganism prior to subsequent testing. Culture of microorganisms is
expensive and
time consuming and can also suffer from contamination or overgrowth by less
fastidious
microorganisms. The techniques are also relatively crude in that many tests
must be
done on the same sample in order to reach definitive diagnosis. Most
microorganisms
can not be readily grown in known media, and hence they fall below levels of
detection
when a typical mixed population of different species of microorganism is
present in the
wild or in association with higher organisms.
Other methods for the detection and identification of pathogenic
microorganisms
are based on the serological approach in which antibodies are produced in
response to
infection with the microorganism. Meningococci, for example, are classifiable
on the
basis of the structural differences in their capsular polysaccharides. These
have different
antigenicities, allowing five major serogroups to be determined, (A, B, C, Y
and W-135).
Enzyme Linked Immunosorbent Assays (ELISA) or Radio lmmuno Assay (RIA) can
assess the production of such antibodies. Both these methods detect the
presence of
specific antibodies produced by the host animal during the course of
infection. These
methods suffer the drawback in that it takes some time for an antibody to be
produced by
the host animal, thus very early infections are often missed. In addition, the
use of such
assays cannot reliably differentiate between past and active infection.
More recently, there has been much interest in the use of molecular methods
for
the diagnosis of infectious disease. These methods offer sensitive and
specific detection
of pathogenic microorganisms. Examples of such methods include the "branched
DNA"
signal detection system. This method is an example that uses the sandwich
hybridization principle (Urdea MS et al. Branched DNA amplification multimers
for the
sensitive, direct detection of human HIV and hepatitis viruses. Nucleic Acids
Symp Ser.
1991; (24):197-200).
Another method for the detection and classification of bacteria is the
amplification
of 16S ribosomal RNA sequences. 16S rRNA has been reported to be a suitable
target
for use in PCR amplification assays for the detection of bacterial species in
a variety of
clinical or environmental samples and has frequently been used to identify
various
specific microorganisms because 16S rRNA genes show species-specific
polymorphisms (Cloud, J. L., H. Neal, R. Rosenberry, C. Y. Turenne, M. Jama,
D. R.
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Hillyard, and K. C. Carroll. 2002. 'J. Clin. Microbiol. 40:400-406). However,
pure culture
of bacteria are required and after PCR amplification the sample still has to
be sequenced
or hybridized to a micro-array type device to determine the species (Fukushima
M,
Kakinuma K, Hayashi H, Nagai H, Ito K, Kawaguchi R. J Clin Microbiol. 2003
Jun;
41(6):2605-15). Such methods are expensive, time consuming and labour
intensive.
Although the genomes of most microorganisms have been determined by ,
sequence analysis, there are still problems in obtaining specific probes or
primers to
detect microorganisms of interest. As genomes contain four bases, often it is
difficult to
prepare sufficient degenerate primers or probes that will be specific for a
given
microorganism. Another potential problem is that the rights to use some
important genes
or genomes of particular microorganisms are owned through patent rights. This
ownership can prevent or delay competing detection assays coming to market.
There is
a need for new nucleic acids that can be used as markers for specific
microorganisms.
The present inventors have obtained modified nucleic acids for numerous
microorganisms that are microbial specific and can be used for detecting
microorganisms.
Summary of Invention
In one aspect, the present invention provides a derivative or modified nucleic
acid
for Hepatitis C virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 76 in Sequence Listing #51, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Acinetobacter sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #1, parts thereof comprising
at least
about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto under
stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Bacillus sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #2, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
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In another aspect, the present invention provides a derivative or modified
nucleic
acid for Bacteroides sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #3, parts thereof comprising
at least
about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto under
stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Bartonella sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #4, parfs.thereof comprising
at least
about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto under
stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Bordetella sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #5, parts thereof comprising
at least
about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto under
stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Borrelia sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #6, parts thereof comprising
at least
about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto under
stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Brucella sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #7, parts thereof comprising
at least
about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto under
stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Campylobacter sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #8, parts thereof comprising
at least
about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto under
stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Chlamydia sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #9, parts thereof comprising
at least
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about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto under
stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Clostridium sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 39 in Sequence Listing #10, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Cornebacterium sp having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #11, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Escherichia coli having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #12, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid moleculcis capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Ehrlichia sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #13, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Enterococcus sp having a sequence selected from the.group consisting
of
SEQ ID NO: I to SEQ ID NO: 4 in Sequence Listing #14, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
= .
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Fusobacterlum=sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #15, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Haemophilus sp having a sequence selected from the group consisting
of
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SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #16, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
5 acid for Helicobacter sp having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #17, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
10 acid for Legionella sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #18, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Leptospira sp having a sequence selected from the group consisting of
*
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #19, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Listeria sp having a sequence selected from the group consisting of
SEQ ID NO: I to SEQ ID NO: 4 in Sequence Listing #20, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Mycobacterium sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #21, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Mycoplasma sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #22, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
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11
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Neisseria sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #23, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Norcadia sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequerice Listing #24, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Pseudomonas sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #25, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Rickettsia sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #26, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Salmonella sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #27, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Seratia sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #28, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Shigella sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #29, parts thereof
comprising at
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12
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent'conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Staphylococcus sp having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 52 in Sequence Listing #30, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Streptococcus sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 16 in Sequence Listing #31, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Streptomyces having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #32, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Treponema sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #33, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Trophermya sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #34, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Plasmodium sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 60 in Sequence Listing #35, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Aspergillis sp having a sequence selected from the group consisting
of
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13
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #36, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides=a derivative or modified
nucleic
acid for Candida sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 24 in Sequence Listing #37, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Cryptococcus sp having a sequence selected from the group consisting
of
SEQ ID NO: I to SEQ ID NO: 4 in Sequence Listing #38, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing,
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Paracoccidioides sp having a sequence selected from the group
consisting of
SEQ ID NO: I to SEQ ID NO: 4 in Sequence Listing #39, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Rhizopus sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #40, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Francisella sp having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #41, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Vibrio sp having a sequence selected from the group consisting of SEQ
ID NO: 1
to SEQ ID NO: 4 in Sequence Listing #42, parts thereof comprising at least
about 20
nucleotides, and nucleic acid molecules capable of hybridizing thereto under
stringent
conditions.
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14
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Yersinia sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #43, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for JC polyomavirus having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ.ID NO: 4 in Sequence Listing #44, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Andes virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #46, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for hepatitis virus having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 24 in Sequence Listing #52, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Human Immunodeficiency virus (HIV) having a sequence selected from
the
group consisting of SEQ ID NO: 1 to SEQ ID NO: 128 in Sequence Listing #53,
parts
thereof comprising at least about 20 nucleotides, and nucleic acid molecules
capable of
hybridizing thereto under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Influenza virus having a sequence selected from the group consisting
of
SEQ ID NO: I to SEQ ID NO: 162 in Sequence Listing #54, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative, or modified
nucleic
acid for BK virus having a sequence selected from the group consisting of SEQ
ID NO: 1
to SEQ ID NO: 4 in Sequence Listing #55, parts thereof comprising at least
about 20
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nucleotides, and nucleic acid molecules capable of hybridizing thereto under
stringent
conditions:
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Barmah virus having a sequence selected from the group consisting of
5 SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #56, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Calcivirus virus having a sequence selected from the group consisting
of
10 SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #57, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Colorado tick fever virus having a sequence selected from the group
consisting
15 of SEQ ID NO: 1 to SEQ ID NO: 48 in Sequence Listing #58, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Foot and Mouth virus having a sequence selected from the group
consisting of
SEQ ID NO: I to SEQ ID NO: 28 in Sequence Listing #59, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Hepatitis GB virus having a sequence selected from the group
consisting of
SEQ ID NO: I to SEQ ID NO: 4 in Sequence Listing #60, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Henda virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #61, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Human adenovirus having a sequence selected from the group consisting
of
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16
SEQ ID NO: 1 to SEQ ID NO: 24 in Sequence Listing #62, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions:
In another aspect, the present invention provides a derivative or modified
nucleic
5, acid for Human astrovirus having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #63, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Human bocavirus virus having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #64, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Human coronavirus having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 16 in Sequence Listing #65, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecufes capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Human enterovirus virus having a sequence selected from the
group'consisting
of SEQ ID NO: 1 to SEQ ID NO: 16 in Sequence Listing #66, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.-
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Human herpes virus having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 36 in Sequence Listing #67, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions,
In another aspect, the present invention provides a derivative or modified
nubleic
acid for Human metapneumovirus having a sequence selected from the group
consisting
of SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #68, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
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In another aspect, the present invention provides a derivative or modified
nucleic
acid for Human parainfluenzavirus having a sequence selected from the group
consisting
of=SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #69, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Human parechovirus having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #70, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Human rhinovirus having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #71, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present`invention provides a derivative or modified
nucleic
acid for Human respiratory syncytial virus having a sequence selected from the
group
consisting of SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #72, parts
thereof
comprising at least about 20 nucleotides, and nucleic acid molecules capable
of
hybridizing thereto under stringent conditions.
In another aspect, the present inventiori provides a derivative or modified
nucleic
acid for Measles virus having a sequence selected from the group consisting of
SEQ ID NO: I to SEQ ID NO: 4 in Sequence Listing #73, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Mumps virus having a sequence selected from the group consisting of
SEQ ID NO: I to SEQ ID NO: 4 in Sequence Listing #74, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Norovirus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #75, parts thereof comprising
at
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18
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Norwalk virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #76, parts thereof comprising
at
least 'about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention prbvides a derivative or modified
nucleic
acid for Parvovirus B19having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #77, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Poliovirus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #78, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Rabies virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #79, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stririgent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Ross River virus having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #80, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Rotavirus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 124 in Sequence Listing #81, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for SARS coronavirus having a sequence 'selected from the group
consisting of
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19
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #82, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under-stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for TT virus'having a sequence selected from the group consisting of SEQ
ID NO: 1
to SEQ ID NO: 4 in Sequence Listing #83, parts thereof comprising at least
about 20
nucleotides, and nucleic acid molecules capable of hybridizing thereto under
stringent
conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for TTV minivirus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #84, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for West Nile virus having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #85, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Alpha virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #86, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Camel pox virus having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID: NO: 4 in Sequence Listing #87, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Cow Pox virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #88, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
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In another aspect, the present invention provides a derivative or modified
nucleic
acid for Coxiella sp having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #89, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
5 under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Crimean-Congo HF having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #90, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable bf hybridizing
thereto
10 under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Dengue virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 16 in Sequence Listing #91, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
15 under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Eastern Equine Encephalitis virus having a sequence selected from the
group
consisting of SEQ ID NO: 1 to SEQID NO: 4 in Sequence Listing #92, parts
thereof
comprising at least about 20 nucleotides, and nucieic acid molecules capable
of
20 hybridizing thereto under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Ebola virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #93, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Marburg virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #94, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Guanarito virus having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #95, parts thereof comprising
at
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21
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Hanta virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #96, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Hantan virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #97, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Japanese encephalitis virus having a sequence selected from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #97, parts
thereof
comprising at least about 20 nucleotides, and nucleic acid molecules capable
of
hybridizing thereto under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Junin virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #99, parts thereof comprising
at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Lassa virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #100, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present inverition provides a derivative or modified
nucleic
acid for Machupo virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #101, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Monkey pox virus having a sequence selected from the group consisting
of
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22
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #102, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Murray Valley encephalitis virus having a sequence selected from the
group
consisting of SEQ ID NO: I to SEQ ID NO: 4 in Sequence Listing #103, -parts
thereof
comprising at least about 20 nucfeotides, and nucleic acid molecules capable
of
hybridizing thereto under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Nipah virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #104, parts thereof
comprising at
least about 20 nucleotides,'and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Rift Valley Fever virus having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #105, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
'acid for Sabia virus having a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 8 in Sequence Listing #106, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Sin Nombre virus having a sequence selected from the group consisting
of
SEQ ID NO: 1 to SEQ ID NO: 12 in Sequence Listing #107, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present inventi6n provides a derivative or modified
nucleic
acid for Variola major virus having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #108, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
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In another aspect, the present invention provides a derivative or modified
nucleic
acid for Variola minor virus having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #109, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Venezuelan equine encephalitis virUs having a sequence selected from
the
group consisting of SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #110,
parts
thereof comprising at least about 20 nucleotides, and nucleic acid molecules
capable of
hybridizing thereto under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Western equine encephalitis virus having a sequence selected from the
group
consisting of SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #111, parts
thereof
comprising at least about 20 nucleotides, and nurleic acid molecules capable
of
hybridizing thereto under stringent conditions.
In another aspect, the present invention provides a derivative or modified
nucleic
acid for Yellow Fever virus having a sequence selected from the group
consisting of
SEQ ID NO: 1 to SEQ ID NO: 4 in Sequence Listing #112, parts thereof
comprising at
least about 20 nucleotides, and nucleic acid molecules capable of hybridizing
thereto
under stringent conditions.
The parts of the derivative or microbial nucleic acid can be at least 20, 21,
22,'23,
24, 25, 26, 27, 28, 29, 30, 40, 50, 60 70, 80, 90, 100, etc, or more
nucleotides. In some
derivative or microbial nucleic acid, the part thereof may be less than 20
such as 15, 16,
17, 18 or 19, for example.
Derivative microbial nucleic acid can be formed by treating microbial nucleic
acid
with an agent such as bisulphate that modifies cytosine "to uracil. After
amplification of
the derivative nucleic acid, modified microbial nucleic acid is formed having
substantially
the bases adenine, guanine and thymine.
For double stranded DNA which typically contains no methylated cytosines, the
treating step results in two derivative nucleic acids, each containing the
bases adenine,
guanine, thymine and uracil. The two derivative nucleic acids are produced
from the two
single strands of the double stranded DNA. The two derivative nucleic acids
have
substantially no cytosines but still have the same total number of bases and
sequence
length as the original untreated DNA molecule. Importantly, the two
derivatives are not
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24
complimentary to each other and form a top and a bottom strand. One or more of
the
strands can be used to generate a derivative nucleic acid or amplified to
produce the
modified nucleic acid molecule.
Throughout this specification, unless the context requires otherwise, the word
"comprise", or variations such as "comprises" or "comprising", will be
understood to imply
the inclusion of a stated element, integer or step, or group of elements,
integers or steps,
but not the exclusion of any other element, integer or step, or group of
elements, integers
or steps.
Any discussion of documents, acts, materials, devices, articles or the like
which
has been included in the present specification is solely for the purpose of
providing a
context for the present invention. It is not to be taken as an admission that
any or all of
these matters form part of the prior art base or were common general knowledge
in the
field relevant to the present invention as it existed in Australia before the
priority date of
each claim of this specification.
In order that the present invention may be more clearly understood, preferred
embodiments will be described with reference to the following drawings and
examples.
Brief Description of the Drawings
Figure 1 shows Hepatitis C 1a genome (top strand) sequence (SEQ ID NO: 77 of
Sequence Listing #51).
Figure 2 shows Hepatitis C 1a genome (bottom strand) sequence (SEQ ID NO: 78
of Sequence Listing #51).
Figure 3 shows Hepatitis C 1 a derivative genome (top strand) sequence
(SEQ ID NO: I of Sequence Listing #51).
Figure 4 shows Hepatitis C 1a derivative genome (bottom strand) sequence
(SEQ ID NO: 20 of Sequence Listing #51).
Figure 5 shows Hepatitis C 1a modified genome (top strand) sequence
(SEQ ID NO: 39 .of Sequence Listing #51).
Figure 6 shows Hepatitis C 1a modified genome (bottom strand) sequence
(SEQ ID NO: 58 of Sequence Listing #51).
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Mode(s) for Carrying Out the Invention
Definitions
The term "genomic modification" as used herein means the genomic (or other)
nucleic acid is modified from being comprised of four bases adenine (A),
guanine (G),
5 thymine (T) and cytosine (C) to substantially containing the bases adenine
(A), guanine
(G), thymine (T) but still having substantially the same total number of
bases.
The term "derivative nucleic acid " as used herein means a nucleic acid that
substantially contains the bases A, G, T and U (or some other non-A, G or T
base or
base-like entity) and has substantially the same total number of bases as the
10 corresponding unmodified microbial nucleic acid. Substantially all
cytosines in the'
microbial DNA will have been converted to uracil during treatment with the
agent. It will
be appreciated that altered cytosines, such as by methylation, may not
necessarily be
converted to uracil (or some other non-A, G or T base or base-like entity). As
microbial
nucleic acid typically does not contain methylated cytosine (or other cytosine
alterations)
15 the treated step preferably converts all cytosines. Preferably, cytosine is
modified to
uracil.
The term "modified nucleic acid" as used herein means the resulting nucleic
acid
product obtained after amplifying derivative nucleic acid. Uracil in the
derivative nucleic
acid is then replaced as a thymine (T) during amplification of the derivative
nucleic acid
20 to form the modified nucleic acid molecule. The resulting product has
substantially the
same number of total bases as the corresponding unmodified microbial nucleic
acid but
is substantially made up of a combination of three bases (A, G and T).
The term "modified sequence" as used herein means the resulting nucleic acid
sequence obtained after amplifying derivative nucleic acid to form a modified
nucleic'
25 acid. The resulting modified sequence has substantially the same number of
total bases
as the corresponding unmodified microbial nucleic acid sequence but is
substantially
made up of a combination of three bases (A, G and T).
The term "non-converted sequence" as used herein means the nucleic acid
sequence of the microbial nucleic acid prior to treatment. A non-converted
sequence
typically is the sequence of the naturally occurring microbial nucleic acid.
The term "modifies" as used herein means the conversion of a cytosine to
another nucleotide. Preferably, the agent modifies cytosine to uracii to form
a derivative
nucleic acid.
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26
The term "agent that modifies cytosine" as used herein means an agent that is
capable of converting cytosine to another chemical entity. Preferably, the
agent modifies
cytosine to uracil which is then replaced as a thymine during amplification of
the
derivative nucleic acid. Preferably, the agent used for modifying cytosine is
sodium
bisulfite. Other agents that similarly modify cytosine, but not methylated
cytosine can
also be used in the method of the invention. Examples include, but not limited
to
bisulfite, acetate or citrate. Preferably, the agent is sodium bisulfite, a
reagent, which in
the presence of acidic aqueous conditions, modifies cytosine into uracil.
Sodium bisulfite
(NaHSO3) reacts readily with the 5,6-double bond of cytosine to form a
sulfonated
cytosine reaction intermediate which is susceptible to deamination, and in the
presence
of water gives rise to a uracil sulfite. If necessary, the sulfite group can
be removed
under mild alkaline conditions, resulting in the formation of uracil. Thus,
potentially all
cytosines will be converted to uracils. Any methylated cytosines, however,
cannot be
converted by the modifying reagent due to protection by methylation. It will
be
appreciated that cytosine (or any other base) could be modified by enzymatic
means to
achieve a derivative nucleic acid as taught by the present invention:
There are two broad generic methods by which bases in nucleic acids may be
modified: chemical and enzymatic. Thus, modification for the present invention
can also
be carried out by naturally occurring enzymes, or by yet to be reported
artificially
= 20 constructed or selected enzymes. Chemical treatment, such as bisulphite
methodologies, can convert cytosine to uracil via appropriate chemical steps.
Similarly,
cytosine deaminases, for example, may carry out a conversion to form a
derivative
nucleic acid. The first report on cytosine deaminases to our knowledge is
1932, Schmidt,
G., Z. physiol. Chem., 208, 185; (see also 1950, Wang, T:P., Sable, H.Z.,
Lampen, J.O.,
J. Biol. Chem, 184, 17-28, Enzymatic deamination of cytosines nucleosides). In
this
early work, cytosine deaminase was not obtained free of other nucleo-
deaminases,
however, Wang et al. were able to purify such an activity from yeast and E.
coli. Thus
any enzymatic conversion of cytosine to form a derivative nucleic acid which
ultimately
results in the insertion of a base during the next replication at that
position, that is
different to a cytosine, will yield a modified genome. The chemical and
enzymatic
conversion to yield a derivative followed by a modified genome are applicable
to any
nucleo-base, be it purines or pyrimidines in naturally occurring nucleic acids
of
microorganisms.
The term "modified form of the genome or nucleic acid" as used herein means
that a genome or nucleic acid, whether naturally occurring or synthetic, which
usually
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27
contains the four common bases G, A, T and C, now consists largely of only
three bases,
G, A and T since most or all of the Cs in the genome have been converted to Ts
by
appropriate chemical modification and subsequent amplification procedures. The
modified form of the genome means that relative genomic complexity is reduced
from a
four base foundation towards a three base composition.
The term 'base-like entity' as used herein means an entity that is formed by
modification of cytosine. A base-like entity can be recognised by a DNA
polymerase
during amplification of a derivative nucleic acid and the polymerase causes A,
G or T to
be placed on a newly formed complementary DNA strand at the position opposite
the
base-like entity in the derivate nucleic acid. Typically, the base-like entity
is uracil that
has been modified from cytosine in the corresponding untreated microbial
nucleic acid.
Examples of a base-like entity includes any nucleo-base, be it purine or
pyrimidine.
The term "relative complexity reduction" as used herein relates to probe
length,
namely the increase in average probe length that is required to achieve the
same
specificity and level of hybridization of a probe to a specific locus, under a
given set of
molecular conditions in two genomes of the same size, where the first genome
is "as is"
and consists of the four bases, G, A T and C, whereas the second genome is of
exactly
the same length but some cytosines, (ideally alf cytosines), have been
converted to
thymines. The locus under test is in the same location in the original
unconverted as well
as the converted genome. On average, an 11-mer probe will have a unique
location to
which it will hybridize perfectly in a regular genome of 4,194,304 bases
consisting of the
four bases G, A, T and C, (411 equals 4,194,304). However, once such a regular
genome of 4,194, 304 bases has been converted by bisulfite or other suitable
means,
this converted genome is=now composed of only three bases and is clearly less
complex.-
However the consequence of this decrease in genomic complexity is that our
previously
unique 11-mer probe no longer has a unique site to which it can hybridize
within the
modified genome. There are now many other possible equivalent locations of 11
base
sequences that have arisen de novo as a consequence of the bisulfite
conversion. It will
now require a 14-mer probe to find and hybridize to the original locus.
Although it may
initially appear counter intuitive, one thus requires an increased probe
length to detect
the original location in what is now a modified three base genome, because
more of the
genome looks the same, (it has more similar sequences). Thus the reduced
relative
genomic complexity, (or simplicity of the three base genome), means that one
has to
design longer probes to find the original unique site.
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The term "relative genomic complexity reduction" as used herein can be
measured by increased probe lengths capable of being microbe-specific as
compared
with unmodified DNA. This term also incorporates the type of probe sequences
that are
used in determining the presence of a microorganism. These probes may have non-
conventional backbones, such as those of PNA or LNA or modified additions to a
backbone such as those described in INA. Thus, a genome is considered to have
reduced relative complexity, irrespective of whether the probe has additional
components
such as Intercalating pseudonucleotides, such as in INA. Examples include, but
not
limited to, DNA, RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA),
MNA,
altritol nucleic acid (ANA), hexitol nucleic acid (HNA), intercalating nucleic
acid (INA),
cyciohexanyl nucleic acid (CNA) and mixtures thereof and hybrids thereof, as
well as
phosphorous atom modifications thereof, such as but not limited to
phosphorothioates,
methyl phospholates, phosphoramidites, phosphorodithiates,
phosphoroselenoates,
phosphotriesters and phosphoboranoates. Non-naturally occurring nucleotides
include,
but not limited to the nucleotides comprised within DNA, RNA, PNA, INA, HNA,
MNA,
ANA, LNA, CNA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, a-L-Ribo-LNA, a-L-Xylo-
LNA,
[i-D-Xylo-LNA, a-D-Ribo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-
epi-
Bicyclo-DNA, a-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-
DNA,
Bicyclo[4.3.0]amide-DNA, [3-D-Ribopyranosyl-NA, a-L-Lyxopyranosyl-NA, 2'-R-
RNA, a-L-
RNA or a-D-RNA, [i-D-RNA. In addition non-phosphorous containing compounds may
be used for linking to nucleotides such as but not limited to
methyliminomethyl,
formacetate, thioformacetate and linking groups comprising amides. In
particular nucleic
acids and nucleic acid-analogues may comprise one or more intercalator
pseudonucieotides (IPN). The presence of IPN is not part of the complexity
description
for nucleic acid molecules, nor is the backbone part of that complexity, such
as in PNA.
By'INA' is meant an intercalating nucleic acid in accordance with the teaching
of
WO 03/051901, WO-03/052132, WO 03/052133 and WO 03/052134 (Unest A/S)
incorporated herein by reference. An INA is an oligonucleotide or
oligonucleotide
analogue comprising one or more intercalator pseudonucleotide (IPN) molecules.
By'HNA' is meant nucleic acids as for example described by Van Aetschot et
al.,
1995.
By'MNA' is meant nucleic acids as described by Hossain et al, 1998.
'ANA' refers to nucleic acids described by Allert et al, 1999.
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'LNA' may be any LNA molecule as described in WO.99/14226 (Exiqon),
preferably, LNA is selected from the molecules depicted in the abstract of WO
99/14226.
More preferably, LNA is a nucleic acid as described in Singh et al, 1998,
Koshkin et al,
1998 or Obika et al., 1997.
'PNA' refers to peptide nucleic acids as for example described by Nielsen et
al,
1991.
'Relative complexity reduction' as used herein, does not refer to the order in
which bases occur, such as any mathematical complexity difference between a
sequence that is ATATATATATATAT (SEQ ID NO: ) versus one of the same length
that
is AAAAAAATTTTTTT (SEQ ID NO: ), nor does it refer to the original re-
association
data of relative genome sizes, (and inferentially, genomic complexities),
introduced into
the.scientific literature by Waring, M. & Britten R. J.1966, Science, 154, 791-
794; and
Britten, R.J and Kohne D E., 1968, Science, 161, 529-540, and earlier
references therein
that stem from the Carnegie Institution of Washington Yearbook reports.
'Relative genomic complexity' as used herein refers to an unchanged position
of
bases in two genomes that is accessed by molecular probes (both the original
and
unconverted genomes have bases at invariant positions 1 to n. In the case of
the
3 billion base pair haploid human genome of'a particular human female, the
invariant
positions are defined as being from 1 to n, where n is 3,000,000,000. If in
the sequence
1 to n, the i`h base is a C in the original genome, then the i`" base is a T
in the converted
genome.
The term "genomic nucleic acid" as used herein includes microbial (prokaryote
and single celled eukaryote) RNA, DNA, protein encoding nucleic acid, non-
protein
encoding nucleic acid, and ribosomal gene regions of prokaryotes and single
celled
eukaryotic microorganisms.
The term "microbial genome" as used herein covers chromosomal as well as
extrachromosomal nucleic acids, as well as temporary residents of that genome,
such a
plasmids, bacteriphage and mobile elements in the broadest sense. The "genome"
has
a core component as exemplified by S. galactiae, as well as possibly having
coding and
'non-coding elements that vary between different isolates.
The term "microbial derived DNA" as used herein includes DNA obtained directly
from a microorganism or obtained indirectly by converting microbial RNA to DNA
by any
of the known or suitable method such as reverse transcriptase.
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The term "microorganism" as used herein includes phage, virus, viroid,
bacterium, fungus, alga, protozoan, spirochaete, single cell organism, or any
other
microorganism, no matter how variously classified, such as the Kingdom
Protoctista by
Margulis, L., et a/1990, Handbook of Protoctista, Jones and Bartlett,
Publishers, Boston
5 USA, or microorganisms that are associated with humans, as defined in
Harrisons
Principles of Internal Medicine, 12 th Edition, edited by J D Wilson et al.,
McGraw Hill Inc,
as well as later editions. It also includes all microorganisms described in
association with
human conditions defined in OMIM, Online Mendelian Inheritance in Man,
www.ncbi.gov.
The term "microbial-specific nucleic acid molecule" as used herein means a
10 molecule which has been determined or obtained using the method according
to the
present invention which has one or more sequences specific to a microorganism.
The term "taxonomic level of the microorganism" as used herein includes
family,
genus, species, strain, type, or different populations from the same or
different
geographic or benthic populations. While in the case of bacteria the generally
15 recognized schema, such as; Bacteria, Proteobacteria; Betaproteobacteria;
Neisseriales;
Neisseriaceae; Neisseria is used. Different populations may be polymorphic for
single
nucleotide changes or variation that exists in DNA molecules that exist in an
intracellular
form within a microorganism (plasmids or phagemids), or polymorphic
chromosomal
regions of microorganism genomes such as pathogenicity islands. The fluidity
of =
20 microbial and viral genomes is recognized, and includes the chimeric nature
of viral
genomes, which can be in independent nucleic acid pieces. Hence, newly arising
strains
from re-assortment of genomic regions from different animals .e.g., new human
influenza
strains as chimeras of segments that are picked up from other mammalian or
avian viral
genomes.
25 The term "close sequence similarity" as used herein includes the above
definition
of relative sequence complexity and probe lengths as a measure.
The term "hybridizing under stringent conditions" is used interchangeably with
the
term "capable of hybridizing under stringent conditions" herein to mean that
nucleic acids
may be readily identified by their ability to hybridize under stringent
conditions with all or
30 parts of a modified microbial nucleic acid. By capable of hybridizing under
stringent
conditions it is meant that annealing of nucleic acid occurs under standard
conditions,
e.g., high temperature and/or low salt content, which tend to preclude
hybridization of
noncomplementary nucleotide sequences. An example of a stringent protocol for
hybridization of nucleic acid probes to immobilised DNA (involving 0.1xSSC, 68
C for 2
hours) is described in Maniatis, T., et al., Molecular Clon.ing: A Laboratory
Manual, Cold
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Springs Harbor Laboratory, 1982,.at pages 387-389, although conditions will
vary
depending on the application.
MATERIALS and METHODS
Extraction of DNA
In general, microbial DNA (or viral RNA) can be obtained from any suitable
source. Examples include, but not limited to, cell cultures, broth cultures,
environmental
samples, clinical samples, bodily fluids, liquid samples, solid samples such
as tissue.
Microbial DNA from samples can be obtained by standard procedures. An example
of a
suitable extraction is as follows. The sample of interest is placed in 400 pl
of 7 M
Guanidinium hydrochloride, 5'mM EDTA, 100 mM Tris/HCI pH 6.4, 1% Triton-X-100,
50 mM Proteinase K (Sigma), 100 Ng/mi yeast tRNA. The sample is thoroughly
homogenised with disposable 1.5 ml pestle and left for 48 hours at 600C. After
incubation the, sample is subjected to five freeze/thaw cycles of dry ice for
5
minutes/95 C for 5 minutes. The sample is then vortexed and spun in a
microfuge for
2 minutes to pellet the cell debris. The supernatant is removed into a clean
tube, diluted
to reduce the salt concentration then phenol:chloroform extracted, ethanol
precipitated
and resuspended in 50 pl of 10 mM Tris/0.1 mM EDTA.
Specifically, the DNA extractions from Gram positive and Gram negative
bacteria
grown on standard agar plates (with nutritional requirements specific to each
species)
were performed as follows.
For DNA extraction from Gram Negative bacteria the protocol was as follows:
a) Using a sterile toothpick bacterial colonies were scraped off the culture
plate into a
sterile 1.5 ml centrifuge tube.
b) 180 pl of Guanidinium=thiocyanate extraction buffer (7M Guanidinium
thiocyanate, 5
mM EDTA (pH8.0), 40 mM Tris/Hcl pH 7.6, 1% Triton-X-100) was added and the
sample mixed to resuspend the bacterial colonies.
c) 20 pl (20 mg/mi) Proteinase K was added and the samples were mixed well.
d) Samples were incubated @ 55 C for 3 hours to lyse the cells.
e) 200 pl of water was added to each sample and samples mixed by gentle
pipetting.
f) 400 pl of Phenol/Chloroform/iso-amyl alcohol (25:24:1) was added and the
samples
vortexed for 2 X 15 seconds.
g) The samples were then spun in a microfuge at 14,000 rpm for 4 minutes.
h) The aqueous phase was removed into a clean 1.5 ml centrifuge tube.
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i) 400 pl of Phenol/Chloroform/iso-amyl alcohol (25:24:1) was added and the
samples
vortexed for 2 X 15 seconds.
j) The samples were then spun in a microfuge at 14,000rpm for 4 minutes.
k) The aqueous phase was removed into a clean 1.5 ml centrifuge tube.
I) 800 pl of 100% ethanol was added to each sample, the sample vortexed
briefly then
left at -20 c for 1 hour.
m) The "samples were spun in a microfuge at 14,000 rpm for 4 minutes at 4 C.
n) The DNA pellets were washed with 500 pl of 70% ethanol.
o) The samples were spun in a microfuge at 14,000rpm for 5 minutes at 4 C, the
ethanol was discarded and the pellets were air dried for 5 minutes.
p) Finally the DNA was resuspended in 100 pl of 10 mM Tris/HCI pH 8.0, 1 mM
EDTA
pH 8Ø
q) The DNA concentration and purity were calculated by measuring the
absorbance of
the solution at 230, 260, 280nm.
For DNA extraction from Gram Positive bacteria the protocol was as follows:.
a) Using a sterile toothpick bacterial colonies were scraped off the culture
plate into a
sterile 1.5 ml centrifuge tube.
b) 180 pl of 20 mg/mi Lysozyme (Sigma) and 200 pg of Lysostaphin (Sigma) was
added
to each sample and the samples were mixed gently to resuspend the bacterial
colonies.
c) The samples were incubated at 37 C for 30 minutes to degrade the cell wall.
d) The samples were then processed and the DNA extracted according to the
QlAamp
DNA mini kit protocol for Gram positive bacteria.
DNA extraction from Cytology samples from patients.
a) The sample was shaken vigorously by hand to resuspend any sedimented cells
and
to ensure the homogeneity of the solution.
b) 4 ml of the resuspended cells were transferred to a 15 ml Costar centrifuge
tube.
c) The tubes were centrifuged in a swing-out bucket rotor at 3000 x g for 15
minutes.
d) The supernatant was carefully decanted and discarded without disturbing the
pelleted cellular material.
e) The pelleted cells were resuspended in 200 pl of lysis buffer (100 mM
Tris/HCI
pH 8.0, 2 mM EDTA pH 8.0, 0.5% SDS, 0.5% Triton-X-100) and mixed well until
the
solution was homogeneous.
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f) 80 pl of the sample was transferred to a 96 well sample preparation plate
g) 20 pl of Proteinase K was added and. the solution incubated at 55 C for 1
hour (this
procedure results in cell lysis)
DNA extraction from urine samples
DNA was extracted from a starting volume of 1 ml of urine according to the
QlAamp UltraSensTM Virus Handbook.
Bisulfite treatment of DNA samples
Bisulfite treatment was carried out according the MethylEasyTM High Throughput
DNA bisulfite modification kit (Human Genetic Signatures, Australia) see also
below..
Surprisingly, it has been found by the present inventors that there is no need
to
separate the microbial DNA from other sources of nucleic acids, for,example
when there
is microbial DNA in a sample of human cells. The treatment step can be used
for an vast
mixture of different DNA types and yet a microbial-specific nucleic acid can
be stifl
identified by the present invention. It is estimated that the limits of
detection in a complex
DNA mixtures are that of the limits of standard PCR detection which can be
down to a
single copy of a target nucleic acid molecule.
Samples
'20 Any suitable sample can be used for the present invention. Examples
include,
but not limited to, microbial cultures, clinical samples, veterinary samples,
biological
fluids, tissue culture samples, environmental samples, water samples,
effluent. As the
present invention is adaptable for detecting any microorganism, this list
should not be
considered as exhaustive.
Kits
The present invention can be implemented in the form of various kits, or
combination of kits and instantiated in terms of manual, semi automated or
fully robotic
platforms. In a preferred form, the MethyEasyTM or HighThroughput MethylEasyTM
kits
(Human Genetic Signatures Pty Ltd, Australia) allow conversion of nucleic
acids in 96 or
384 plates using a robotic platform such as EpMotion.
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Bisulfite treatment
An exemplary protocol for effective bisulfite treatment of nucleic acid is set
out
below. The protocol results in retaining substantially all DNA treated. This
method is
also referred to herein as the Human Genetic Signatures (HGS) method. It will
be
appreciated that the volumes or amounts of sample or reagents can be varied.
Preferred method for bisulfite treatment can be found in US 10/428310 or
PCT/AU2004/000549 incorporated herein by reference.
To 2 pg of DNA, which can be pre-digested with suitable restriction enzymes if
so
desired, 2 pl (1/10 volume) of 3 M NaOH (6g in 50 ml water, freshly made) was
added in
a final volume of 20 pl. This step denatures the double stranded DNA molecules
into.a
single stranded form, since the bisulfite reagent preferably reacts with
single stranded
molecules. The mixture was incubated at 37 C for 15 minutes. Incubation at
temperatures above room temperature can be used to improve the efficiency of
denaturation.
After the incubation, 208 pl 2 M Sodium Metabisulfite (7.6 g in 20 ml water
with
416 ml 10 N NaOH; BDH AnalaR #10356.4D; freshly made) and 12 pl of 10 mM
Quinol
(0.055 g in 50 ml water, BDH AnaIR #103122E; freshly made) were added in
succession.
Quinol is a reducing agent and helps to reduce oxidation of the reagents.
Other reducing
agents can also be used, for example, dithiothreitol (DTT), mercaptoethanol,
quinone
(hydroquinone), or other suitable reducing agents. The sample was overlaid
with 200 pl
of mineral= oil. The overlaying of mineral oil prevents evaporation and
oxidation of the
reagents but is not essential. The sample was then incubated overnight at 55
C.
Alternatively the samples can be cycled in a thermal cycler as follows:
incubate for about
4 hours or overnight as follows: Step 1, 55 C / 2 hr cycled in PCR machine;
Step 2, 95 C
/ 2 min. Step 1 can be performed at any temperature from about 37 C to about
90 C and
can vary in length from 5 minutes to 8 hours. Step 2 can be performed at any
temperature from about 70 C to about 99 C and can vary in length from about 1
second
to 60 minutes, or longer.
After the treatment with Sodium Metabisulfite, the oil was removed, and 1 pl
tRNA
(20 mg/mI) or 2 pl glycogen were added if the DNA concentration was low. These
additives are optional and can be used to improve the yield of DNA obtained by
co-
precipitating with the target DNA especially when the DNA is present at low
concentrations. The use of additives as carrier for more efficient
precipitation of nucleic
acids is generally desired when the amount nucleic acid is <0.5 pg.
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An isopropanol cleanup treatment was performed as follows: ' 800 pl of water
were added to the sample, mixed and then I ml isopropanol was added. The water
or
buffer reduces the concentration of the bisulfite salt in the reaction vessel
to a level at
which the salt will not precipitate along with the target nucleic acid of
interest. The
5 dilution is generally about 1/4 to 1/1000 so long as the salt concentration
is diluted below
a desired range, as disclosed herein.
The sample was mixed again and left at 4 C for a minimum of 5 minutes. The
sample was spun in a microfuge for 10-15 minutes and the pellet was washed 2x
with
70% ETOH, vortexing each time. This washing treatment removes any residual
salts
10 that precipitated with the nucleic acids.
The pellet was allowed to dry and then resuspended in a suitable volume of TIE
(10 mM Tris/0.1 mM EDTA) pH 7.0-12.5 such as 50 pf. Buffer at pH 10.5 has been
found to be particularly effective. The sample was incubated at 37 C to 95 C
for 1 min to
96 hr, as needed to suspend the nucleic acids.
15 Another example of bisulfite treatment can be found in WO 2005021778
(incorporated herein by reference) which provides methods and materials for
conversion
of cytosine to uracil. In some embodiments, a nucleic acid, such as gDNA, is
reacted
with bisulfite and a polyamine catalyst, such as a triamine or tetra-amine:
Optionally, the
bisulfite comprises magnesium bisulfite. In other embodiments, a nucleic acid
is reacted
20 with magnesium bisulfite, optionally in the presence of a polyamine
catalyst and/or a
quaternary amine catalyst. Also provided are kits that can be used to carry
out methods
of the invention. It will be appreciated that these methods would also be
suitable for the
present invention in the treating step.
25 Amplification
PCR amplifications were performed in 25 p1 reaction mixtures containing 2 pl
of
bisulfite-treated genomic DNA, using the Promega PCR master mix, 6 ng/pl of
each of
the primers. Strand-specific nested primers are used for amplification. 1 St
round PCR
amplifications were carried out using PCR primers I and 4 (see below).
Following 1st
30 round amplification, 1 ial of the amplified material was transferred to 2d
round PCR
premixes containing PCR primers 2 and 3 and amplified as previously described.
Samples of PCR products were amplified in a ThermoHybaid PX2 thermal cycler
under
the conditions: 1 cycle of 95 C for 4 minutes, followed by 30 cycles of 95 C
for 1 minute,
50 C for 2 minutes and 72 C for 2 minutes; I cycle of 72 C for 10 minutes.
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36
#1 0 ~#4
#2 ~ #3
5. Multiplex amplification
If multiplex amplification is required for detection, the following
methodology can
be carried out.
One pl of bisulfite treated DNA is added to the following components in a 25
pl
reaction volume, xl Qiagen multiplex master mix, 5-100 ng of each 1 st round
INA or
oligonucleotide primer 1.5- 4.0 mM MgSO4, 400 uM of each dNTP and 0.5-2 unit
of the
polymerase mixture. The components are then cycled in a hot lid thermal cycler
as
follows. Typically there can be up to 200 individual primer sequences in each
amplification reaction
Step 1 94 C 15 minute 1 cycle
Step 2 94 C 1 minute
50 C 3 minutes 35 cycles
68 C 3 minutes
Step 3 68 C 10 minutes 1 cycle
A second round amplification is then performed on a 1pl aliquot of the first
round
amplification that is transferred to a second round reaction tube containing
the enzyme
reaction mix and appropriate second round primers. Cycling is then performed
as above.
Primers
Any suitable PCR primers can be used for the present invention. A primer
typically has a complementary sequence to a sequence which will be amplified.
Primers
are typically oligonucleotides but can be oligonucleotide analogues.
Probes
The probe may be any suitable nucleic acid molecule or nucleic acid analogue.
Examples include, but not limited to, DNA, RNA, locked nucleic acid (LNA),
peptide
nucleic acid (PNA), MNA, altritol nucleic acid (ANA), hexitol nucleic acid
(HNA),
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intercalating nucleic acid (INA), cyclohexanyl nucleic acid (CNA) and mixtures
thereof
and hybrids thereof, as well as phosphorous atom modifications thereof, such
as but not
limited to phosphorothioates, methyl phospholates, phosphoramidites,
phosphorodithiates, phosphoroselenoates, phosphotriesters and
phosphoboranoates.
Non-naturally occurring nucleotides include, but not limited to the
nucleotides comprised
within DNA, RNA, PNA, INA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2'-NH)-TNA,
(3'-NH)-TNA, a-L-Ribo-LNA, a-L-Xylo-LNA, P-D-Xylo-LNA, a-D-Ribo-LNA, [3.2.1]-
LNA,
Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, a-Bicyclo-DNA, Tricyclo-
DNA,
Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, [3-D-
Ribopyranosyl-
NA, a-L-Lyxopyranosyl-NA, 2'-R-RNA, a-L-RNA or a-D-RNA, R-D-RNA. In addition
non-
phosphorous containing compounds may be used for linking to nucleotides such
as but
not limited to methyliminomethyl, formacetate, thioformacetate and linking
groups
comprising amides. In particular nucleic acids and nucleic acid analogues may
comprise
one or more intercalator pseudonucleotides.
Preferably, the probes are DNA or DNA oligonucleotides containing one or more
internal IPNs forming INA.
Electrophoresis
Electrophoresis of samples was performed according to the E-gel system user
guide (www.invitrogen.doc).
Detection methods
Numerous possible detection systems exist to'determine the status of the
desired
sample. It will be appreciated that any known system or method for detecting
nucleic
acid molecules could be used for the present invention. Detection systems
include, but
not limited to:
1. Hybridization of appropriately labelled DNA to a micro-array type device
which
could select for 10->200,000 individual components. The arrays could be
composed of either INAs, PNAs or nucleotide or modified nucleotides arrays
onto
any suitable solid surface such as glass, plastic, mica, nylon , bead,
magnetic
bead, fluorescent bead or membrane;
II. Southern blot type detection systems;
Ill. Standard PCR detection systems such as agarose gel, fluorescent read outs
such as Genescan analysis. Sandwich hybridisation assays, DNA staining
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reagents such as ethidium bromide, Syber green, antibody detection, ELISA
plate reader type devices, fluorimeter devices;
IV. Real-Time PCR quantitation of specific or multiple genomic amplified
fragments
or any variation on that.
V. Any of the detection systems outlined in the WO 2004/065625 such as
fluorescent beads, enzyme conjugates, radioactive beads and the like;
VI. Any other detection system utilizing an amplification step such as ligase
chain
reaction or Isothermal DNA amplification technologies such as Strand
Displacement Amplification (SDA).
VII. Multi-photon detection systems.
VIII. Electrophoresis and visualisation in gels.
IX. Any detection plafform used or could be used to detect nucleic acid.
Intercalating nucleic acids
Intercalating nucleic acids (INA) are non-naturally occurring polynucleotides
which can hybridize to nucleic acids (DNA and RNA) with sequence specificity.
INA are
candidates as alternatives/substitutes to nucleic acid probes in probe-based
hybridization
assays because they exhibit several desirable properties. INA are
polymers'which
hybridize to nucleic acids to form hybrids which are more thermodynamically
stable than
a corresponding naturally occurring nucleic acid/nucleic acid complex. They
are not
substrates for the enzymes which are known to degrade peptides or nucleic
acids.
Therefore, INA should be more stable in biological samples, as well as, have a
longer
shelf-life than naturally occurring nucleic acid fragments. Unlike nucleic
acid
hybridization which is very dependent on ionic strength, the hybridization of
an INA with a
nucleic acid is fairly independent of ionic strength and is favoured at low
ionic strength
under conditions which strongly disfavour the hybridization of naturally
occurring nucleic
acid to nucleic acid. The binding strength of INA is dependent on the number
of
intercalating groups engineered into the molecule as well as the usual
interactions from
hydrogen bonding between bases stacked in a specific fashion in a double
stranded
structure. Sequence discrimination is more efficient for INA recognizing DNA
than for
DNA recognizing DNA.
Preferably, the INA is the phosphoramidite of (S)-1 -0-(4,4'-
dimethoxytriphenylmethyl)-3-0-(1 -pyrenylmethyl)-glycerol.
INA are synthesized by adaptation of standard oligonucleotide synthesis
procedures in a format which is commercially available. Full definition of INA
and their
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synthesis can be found in WO 03/051901, WO 03%052132, WO 03/052133 and
WO 03/052134 (Unest A/S, assigned to Human Genetic Signatures Pty Ltd,
Australia)
incorporated herein by reference.
There are indeed many differences between INA probes and standard nucleic
acid probes. These differences can be conveniently broken down into
biological,
structural, and physico-chemical differences. As discussed above and below,
these
biological, structural, and physico-chemical differences may lead to
unpredictable results
when attempting to use INA probes in applications were nucleic acids have
typically
been employed. This non-equivalency of differing compositions is often
observed in the
chemical arts.
With regard to biological differences, nucieic acids are biological materials
that
play a central role in the life of living species as agents of genetic
transmission and
expression. Their in vivo properties are fairly well understood. INA, however,
is a
recently developed totally artificial molecule, conceived in the minds of
chemists and
made using synthetic organic chemistry. It has no known biological function.
Structurally, INA also differs dramatically from nucleic acids. Although both
can
employ common nucleobases (A, C, G, T, and U), the composition of these
molecules is
structurally diverse. The backbones of RNA, DNA and INA are composed of
repeating
phosphodiester ribose and 2-deoxyribose units. INA differ from DNA or RNA in
having
one or more large flat molecules attached via a linker molecule(s) to the
polymer. The
flat molecules intercalate between bases in the complementary DNA stand
opposite the
INA in a double stranded structure.
The physico/chernical differences between INA and DNA or RNA are also
substantial. INA binds to complementary DNA more rapidly than nucleic acid
probes
bind to the same target sequence. Unlike DNA or RNA fragments, INA bind poorly
to
RNA unless the intercalating groups are located in terminal positions. Because
of the
strong interactions between the intercalating groups and bases on the
complementary
DNA strand, the stability of the INA/DNA complex is higher than that of an
anaiogous
DNA/DNA or RNA/DNA complex.
Unlike other nucleic acids such as DNA or RNA fragments or PNA, INA do not
exhibit self aggregation or binding properties.
As INA hybridize to nucleic acids with sequence specificity, INA are useful
candidates for developing probe-based assays and are particularly adapted for
kits and
screening assays. INA probes, however, are riot the equivalent of nucleic acid
probes.
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Consequently, any method, kits or compositions which could improve the
specificity,
sensitivity and reliability of probe-based assays would be useful in the
detection, analysis
and quantitation of DNA containing samples. INA have the necessary properties
for this
purpose.
RESULTS
To demonstrate the present invention, derivative and simplified nucleic acid
for
Hepatitis C 1 a are shown in Figures 2 to 6. Top and bottom strands of
Hepatitis C 1 a
native genome are shown in Figures 1 and 2, respectively. Figure 3 and 4 show
derivative nucleic acid where all cytosines have been replaced by uracils in
the top and
bottom strands, respectively. Figure 5 and 6 show modified nucleic acid where
all uracils
in the derivative nucleic acid have been replaced by thymines to form modified
Hepatitis
C 1 a nucleic acid of top and bottom strands, respectively.
As can be seen from Figures 2 to 6, essentially four new artificial genomes
have
been created for Hepatitis C 1 a which can be used for detection or to obtain
suitable
targets. The derivative and modified nucleic acid do not exist in nature but
are typically
generated by bisulphite treatment (derivative) and amplification (modified).
The invention therefore is directed at novel nucleic acid molecules generated
from naturally occurring microbial nucleic acid which has novel and desirable
uses.
Table 1 shows the list of derivative and modified microbial nucleic acid
sequences according to the invention that are provided in accompanying
Sequence
Listings (numbered in numerical order). As the size and total number of
sequences are
extremely large, paper copies have not been provided in the present
specification. All
sequences are however, incorporated herein by reference.
Table 1
Sequence Microorganism Sequence SEQ ID
Listing# NO:
1 Acinetobacter Acinetobacter U* I
Acinetobacter T* 2
Acinetobacter U RC* 3
Acinetobacter T RC* 4
2 Bacillus Bacillus Anthracis U I
Bacillus Anthracis T 2
Bacillus Anthracis U RC 3
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Bacillus Anthracis T RC 4
Bacllus Cereus U 5
Bacflus Cereus T 6
Baclius Cereus U RC 7
Bacllus Cereus T RC 8
Bacillus Subtilis U 9
Bacillus Subtilis T 10
Bacillus Subtilis U RC 11
Bacillus Subtilis T RC 12
3 Bacteriodes Bacteriodes fragilis U 1
Bacteriodes fragilis T 2
Bacteriodes fragilis U RC 3
Bacteriodes fragilis T RC 4
4 Bartonella Bartonella henselae U 1
Bartonella henselae T 2
Bartonella henselae U RC 3
Bartonella henselae T RC 4
Bartonella quintana U 5
Bartonella quintana T 6
Bartonella quintana U RC 7
Bartonella quintana T RC 8
Bordetella Bordetelia pertussis U 1
Bordetella pertussis T 2
Bordetella pertussis U RC 3
Bordetella pertussis T RC 4
6 Borrelia Borrelia burgdorferi U 1
Borrelia burgdorferi T 2
Borrelia burgdorferi U RC 3
Borrelia burgdorferi T RC 4
7 Brucella Brucella melitensis Chr1 U I
Brucella melitensis Chr1 T 2
Brucella melitensis Chr1 U RC 3
Brucella melitensis Chr1 TRC 4
Brucella melitensis Chr2 U 5
Brucella melitensis Chr2 T 6
Brucella melitensis Chr2 U RC 7
Brucella melitensis Chr2 T RC 8
8 Campylobacter Cam lobacter 'e'uni U I
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Cam pylobacter jejuni T 2
Campylobacterjejuni U RC 3
Cam lobacter jejuni T RC 4
9 Chlamydia Chlamydia Pneumoniae U 1
Chlamydia Pneumoniae T 2
Chiamydia Pneumoniae U RC 3
Chiamydia Pneumoniae T RC 4
Chlamydia trachomatis U 5
Chlamydia trachomatis T 6
Chlamydia trachomatis U RC 7
Chlam dia trachomatis T RC 8
Clostridium Ciostridium botulinum Toxin A U I
Clostridium botulinum Toxin A T 2
Clostridium botulinum Toxin A U RC 3
Clostridium botulinum Toxin A T RC 4
Clostridium botulinum Toxin B U 5
Clostridium botulinum Toxin B T 6
Clostridium botulinum Toxin B U RC 7
Clostridium botulinum Toxin B T RC 8
Clostridium botulinum Toxin C U 9
Clostridium botulinum Toxin C T 10
Clostridium botUlinum Toxin C U RC 11
Clostridium botulinum Toxin C T RC 12
Clostridium botulinum Toxin D U 13
Clostridium botulinum Toxin D T 14
Clostridium botulinum Toxin D U RC 15
Clostridium botulinum Toxin D T RC 16
Clostridium botulinum Toxin E U 17
Clostridium botulinum Toxin E T 18
Clostridium botulinum Toxin E F RC 19
Clostridium botulinum Toxin E T RC 20
Clostridium botulinum Toxin F U 21
Clostridium botulinum Toxin F T 22
Clostridium botulinum Toxin F U RC 23
Clostridium botulinum Toxin F T RC 24
Clostridium dififcile U 25
Clostridium dififcile T 26
Clostridium dififcile U RC 27
Clostridium dififcile T RC 28
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Clostridium perfringens U 29
Clostridium perfringens T 30
Clostridium perfringens U RC 31
Clostridium perfringens T RC 32
Clostridium tetani U 33
Clostridium tetani T 34
Clostridium tetani U RC 35
Clostridium tetani T RC 36
Clostridium botulinum U 37
Clostridium botulinum T 38
Clostridium botulinum U RC 39
Clostridium botulinum T RC 40
11 Corynebacterium Corynebacterium diptheriae U I
Corynebacterium diptheriae T 2
Corynebacterium diptheriae U RC 3
Corynebacterium diptheriae T RC 4
Corynebacterium jeikelum U 5
Corynebacterium jeikeium T 6
Corynebacterium jeikeium U RC 7
Cor nebacterium jeikeium T RC 8
12 Ecoli Ecoli U 1
Ecoli T 2
Ecoli U RC 3
Ecoli T RC 4
13 Ehrlichia Ehrlichia chaffeensis U I
Ehrlichia chaffeensis T 2
Ehrlichia chaffeensis U RC 3
Ehrlichia chaffeensis T RC 4
14 Enterococcus Enterococcus faecalis U 1
Enterococcus faecalis T 2
Enterococcus faecalis U RC 3
Enterococcus faecalis T RC 4
15 Fusobacterium Fusobacterium nucleatum U I
Fusobacterium nucleatum T 2
Fusobacterium nucleatum U RC 3.
Fusobacterium nucleatum T RC 4
16 Haemophilus Haemophilus ducreyi U 1
Haemophilus ducreyi T 2
Haemophilus ducreyi U RC 3
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Haemophilus ducreyi T RC 4
Haemophilus influenzae U 5
Haemophilus influenzae T 6
Haemophilus influenzae U RC 7
Haemophilus influenzae T RC 817 Helicobacter Helicobacter pylori U 1
Helicobacter pylori T 2
Helicobacter pylori U RC 3
Helicobacter lori T RC 4
18 Legionella Legionella pneumophila U 1
Legionella pneumophila T 2
~
Legionella pneumophila U RC 3
Le ionella pneumophila T RC 4
19 Leptospira Leptospira interrogans Chr I U 1
Leptospira interrogans Chr I T 2
Leptospira interrogans Chr I U RC 3
Leptospira interrogans Chr I T RC 4
Leptospira interrogans Chr II U 5
Leptospira interrogans Chr II T 6
Leptospira interrogans Chr II U RC 7
Le tos ira interrogans Chr II T RC 8
20 Listeria Listeria monocytogenes U 1
Listeria monocytogenes T 2
Listeria monocytogenes U RG 3
Listeria monoc o enes T RG 4
21 Mycobacterium Mycobacterium avium U I
Mycobacterium avium T 2
Mycobacterium avium U RC 3
Mycobacterium avium T RC 4
Mycobacterium leprae U 5
Mycobacterium leprae T 6
Mycobacterium leprae U RC 7
Mycobacterium leprae T RC 8
Mycobacterium tuberculosis U 9
Mycobacterium tuberculosis T 10
Mycobacterium tuberculosis U RC 11
M cobacterium tuberculosis T RC 12
22 Mycoplasma Mycoplasma pneumoniae U I
M co lasma pneumoniae T 2
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Sequence Microorganism Sequence SEQ ID
Listin # NO:
Mycoplasma pneumoniae U RC 3
M co lasma pneumoniae T RC 4
23 Neisseria Neisseria gonorrhopae U 1
Neisseria gonorrhoeae T 2
Neisseria gonorrhoeae U RC 3
Neisseria gonorrhoeae T RC 4
Neisseria meningitides sero A U 5
Neisseria meningitides sero A T 6
Neisseria meningitides sero A U RC 7
Neisseria meningitides sero A T RC 8
Niesseria meningitidis sero B U 9
Niesseria meningitidis sero B T 10
Niesseria meningitidis sero B U RC 11
Niesseria meningitidis sero B T RC 12
24 Nocardia Nocardia farcinica U 1
Nocardia farcinica T 2
Nocardia farcinica U RC 3
Nocardia farcinica T RC 4
25 Pseudomonas Pseudomonas aeruginosa U 1
Pseudomonas aeruginosa T 2
Pseudomonas aeruginosa U RC 3
Pseudomonas aeruginosa T RC 4
26 Rickettsia Rickettsia prowazekii U I
Rickettsia prowazekii T 2
Rickettsia prowazekii U RC 3
Rickettsia prowazekii T RC 4
Rickettsia typhi U 5
Rickettsia typhi T 6
Rickettsia typhi U RC 7
Rickettsia typhi T RC 8
Rickettsia conorii U 9
Rickettsia conorii T 10
Rickettsia conorii U RC 11
Rickettsia conorii T RC 12
27 Salmonella Salmonella enterica U I
Salmonella enterica T 2
Salmonella enterica U RC 3
Salmonella enterica T RC 4
Salmonella t himurium U 5
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Sequence Microorganism Sequence SEQ ID
Listin # NO:
Salmonella typhimurium T 6
Salmonella typhimurium U RC 7
Salmonella t himurium T RC 8
28 Serratia Serratia marscesens U I
Serratia marscesens T 2
Serratia marscesens U RC 3
Serratia marscesens T RC 4
29 Shigella Shigella flexneri U 1
Shigelia flexneri T 2
Shigella flexneri U RC 3
Shigella flexneri T RC 4
Shigella boydii U 5
Shigella boydii T 6
Shigeiia boydii U RC 7
Shigella boydii T RC 8
Shigella dysenteriae U 9
Shigella dysenteriae T 10
Shigella dysenteriae U RC 11
Shigella d senteriae T RC 12
30 Staphylococcus Staphylococcus aureus U 1
Staphylococcus aureus T 2
Staphylococcus aureus U RC 3
Staphylococcus aureus T RC 4
Staphylococcus epidermidis U 5
Staphylococcus epidermidis T 6
Staphylococcus epidermidis U RC 7
Staphylococcus epidermidis T RC 8
Staphylococcus haemolyticus U 9
Staphylococcus haemolyticus T 10
Staphylococcus haemolyticus U RC 11
Staphylococcus haemolyticus T RC 12
Staphylococcus 'enterotoxin A U 13
Staphylococcus enterotoxin A T 14
Staphylococcus enterotoxin A U RC 15
Staphylococcus enterotoxin AT RC 16
Staphylococcus enterotoxin B U 17
Staphylococcus enterotoxin B T 18
Staphylococcus enterotoxin A B RC 19
Sta h lococcus enterotoxin A B RC 20
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Staphylococcus enterotoxin C'U 21
Staphylococcus enterotoxin C T 22
Staphylococcus enterotoxin C U RC 23
Staphylococcus enterotoxin C T RC 24
Staphylococcus enterotoxin Cl U 25
Staphylococcus enterotoxin Cl T 26
Staphylococcus enterotoxin Cl U RC 27
Staphylococcus enterotoxin Cl T RC 28
Staphylococcus enterotoxin C3 U 29
Staphylococcus enterotoxin C3 T 30
Staphylococcus enterotoxin C3 U RC 31
Staphylococcus enterotoxin C3 T RC 32
Staphylococcus enterotoxin D U 33
Staphylococcus enterotoxin D T 34
Staphylococcus enterotoxin D U RC 35
Staphylococcus enterotoxin D T RC 36
Staphylococcus enterotoxin E U 37
Staphylococcus enterotoxin E T 38
Staphylococcus enterotoxin E U RC 39
Staphylococcus enterotoxin E T RC 40
Staphylococcus enterotoxin G U 41
Staphylococcus enterotoxin G T 42
Staphylococcus enterotoxin G U RC 43
Staphylococcus enterotoxin G T RC 44
Staphylococcus enterotoxin H U 45
Staphylococcus enterotoxin H T 46
Staphylococcus enterotoxin H U RC 47
Staphylococcus enterotoxin H T RC 48
Staphylococcus enterotoxin J&R U 49
Staphylococcus enterotoxin J&R T 50
Staphylococcus enterotoxin J&R U RC 51
Sta h lococcus enterotoxin J&R T RC 52
31 Streptococcus Streptococcus agalactiae U 1
Streptococcus agalactiae T 2
Streptococcus agalactiae U RC 3
Streptococcus agalactiae T RC 4
Streptococcus mutans U 5
Streptococcus mutans T 6
Streptococcus mutans U RC 7
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Streptococcus mutans T RC 8
Streptococcus pneumonipe U 9
Streptococcus pneumoniae T 10
Streptococcus pneumoniae U RC 11
Streptococcus pneumoniae T RC 12
Streptococcus pyrogene U 13
Streptococcus pyrogene T 14
Streptococcus pyrogene U RC 15
Stre tococcus pyrogene T RC 16
32 Streptomyces Streptomyces coelicolor U I
Streptomyces coelicolor T 2
Streptomyces coelicolor U RC 3
Stre tom ces coelicolor T RC 4
33 Treponema Treponema pallidumU I
Treponema pallidumT 2
Treponema pallidumU RC 3
Treponema pallidumT RC -4
34 Tropheryma Tropheryma whippeiii U I
Tropheryma whippelii T 2
Tropheryma whippelii U RC 3
Tro her ma whi eiii T RC 4
35 Plasmodium Plasmodium falciparum mitochondrion U 1
Plasmodium falciparum mitochondrion T 2
Plasmodium falciparum mitochondrion U RC 3
Plasmodium falciparum mitochondrion T RC 4
Plasmodium falciparum Chr1 U 5
Plasmodium falciparum Chr1 T 6
Plasmodium falciparum Chr1 U RC 7
Plasmodium falciparum Chr1 T RC 8
Plasmodium falciparum Chr2 U 9
Plasmodium falciparum Chr2 T 10
Plasmodium faiciparum Chr2 U RC 11
Plasmodium falciparum Chr2 T RC 12
Plasmodium falciparum Chr3 U 13
Plasmodium falciparum Chr3 T 14
Plasmodium faiciparum Chr3 U RC 15
Plasmodium, falciparum Chr3 T RC 16.
Plasmodium falciparum Chr4 U 17
Plasmodium falciparum Chr4 T 18
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Plasmodiu.m falciparum Chr4 U RC 19
Plasmodium falciparum Chr4 T RC 20
Plasmodium falciparum Chr5 U 21
Plasmodium falciparum Chr5 T 22
Plasmodium falciparum Chr5 U RC 23
Plasmodium falciparum Chr5 T RC 24
Plasmodium falciparum Chr6 U 25
Plasmodium falciparum Chr6 T 26
Plasmodium faiciparum Chr6 U RC 27
Plasmodium falciparum Chr6 T RC 28
Plasmodium falciparum Chr7 U 29
Plasmodium falciparum Chr7 T 30
Plasmodium faiciparum Chr7 U RC 31
Plasmodium falciparum Chr7 T RC 32
Plasmodium falciparum Chr8 U 33
Plasmodium faiciparum Chr8 T 34
Plasmodium falciparum Chr8 U RC 35
Plasmodium falciparum Chr8 T RC 36
Plasmodium falciparum Chr9 U 37
Plasmodium falciparum Chr9 T 38
Plasmodium falciparum Chr9 U RC 39
Plasmodium falciparum Chr9 T RC 40
Plasmodium falciparum ChrlO U 41
Plasmodium falciparum Chr10 T 42
Plasmodium faiciparum Chr10 U RC 43
Plasmodium falciparum ChrlO T RC 44
Plasmodium falciparum Chr11 U 45
Plasmodiurrm falciparum Chr11 T 46
Plasmodium falciparum Chr11 U RC 47
Plasmodium falciparum Chr11 T RC 48
Plasmodium falciparum Chr12 U 49
Plasmodium falciparum Chr12 T 50
Plasmodium faiciparum Chr12 U RC 51
Plasmodium falciparum Chr12 T RC 52
Plasmodium falciparum Chrl3 U 53
Plasmodium faiciparum Chr13 T 54
Plasmodium falciparum Chr13 U RC 55
Plasmodium falciparum Chr13 T RC 56
Plasmodium faici arum Chr14 U 57
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Plasmodium falciparum Chr14 T 58
Plasmodium faiciparum Chr14 U RC 59
Plasmodium falci arurn Ch=r14 T RC 60
36 Aspergillus Aspergillus niger mitochondrion U I
Aspergillus niger mitochondrion T 2
Aspergillus niger mitochondrion U RC 3
Aspergillus niger mitochondrion T RC 4
Aspergillus tubingensis mito U 5
Aspergillus tubingensis mito T 6
Aspergillus tubingensis mito U RC 7
As er illus tubingensis mito T RC 8
37 Candida albicans mito U 1
Candida albicans mito T 2
Candida albicans mito U RC 3
Candida albicans mito T RC 4
Candida glabrate mito U 5
Candida glabrate mito T 6
Candida glabrate mito U RC 7
Candida glabrate mito T RC 8
Candida metapsilosis mito U 9
Candida metapsilosis mito T 10
Candida metapsilosis mito U RC 11
Candida metapsilosis mito T RC 12
Candida orthopsilosis mito U 13
Candida orthopsilosis mito T 14
Candida orthopsilosis mito U RC 15
Candida orthopsilosis mito T RC 16
Candida parapsilosis mito U 17
Candida parapsilosis mito T 18
Candida parapsilosis mito U RC 19
Candida parapsilosis mito T RC 20
Candida stellata mito U 21
Candida stellata mito T 22
Candida stellata mito U RC 23
Candida stellata mito T RC 24
38 Cryptococcus Cryptococcus neoformans var U 1
Cryptococcus neoformans var T 2
Cryptococcus neoformans var U RC 3
Qryptococcus neoformans var T RC 4
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Sequence Microorganism Sequence SEQ ID
Listin # NO:
39 Paracoccidioides Paracoccidioides brasiliensis mito U I
Paracoccidioides brasiliensis mito T 2
Paracoccidioides brasiliensis mito U RC 3
Paracoccidioides brasiliensis mito T RC 4
40 Rhizopus Rhizopus oryzae mitochondrion U 1
Rhizopus oryzae mitochondrion T 2
Rhizopus oryzae mitochondrion U RC 3
Rhizopus or zae mitochondrion T RC 4
41 Francisella Franciselia tularensis U 1
Francisella tularensis T 2
Francisella tularensis U RC 3
Francisella tularensis T RC 4
42 Vibrio Vibrio cholerae chromosoame II U 1
Vibrio cholerae chromosoame 1.1 T 2
Vibrio cholerae chromosoame II U RC 3
Vibrio cholerae chromosoame II T RC 4
43 Yersinia Yersinia pestis U 1
Yersinia pestis T 2
Yersinia pestis U RC 3
Yersinia p estis T RC 4
44 JC polyomavirus JC polyomavirus U I
JC polyomavirus T 2
JC polyomavirus U RC 3
JC Oal omavirus T RC 4
45 1
2
3
4
46 Andes virus Andes virus segment L U 1
Andes virus segment L T 2
Andes virus segment L U RC 3
Andes virus segment L T RC 4
Andes virus segment M U 5
Andes virus segment M T 6
Andes virus segment M U RC 7
Andes virus segment M T RC 8
Andes virus segment S U . 9
Andes virus segment S T 10
Andes virus segment S U RC 11
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Andes virus segment S T RC 12
51 Hepatitis C virus Hepatitis C 1 a T 1
Hepatitis C 2b T 2
Hepatitis C I b/2k T 3
Hepatitis C 1 c T 4
Hepatitis C 2a T 5
Hepatitis C 2b T 6
Hepatitis C 2C T 7
Hepatitis C 2K T 8
Hepatitis C 3a T 9
Hepatitis C 3B T 10
Hepatitis C 3k T 11
Hepatitis C 4a T 12
Hepatitis C 5a T 13
Hepatitis C 6a T 14
Hepatitis C 6b T 15
Hepatitis C 6g T 16
Hepatitis C 6d T 17
Hepatitis C 6h T 18
Hepatitis C 6k T 19
Hepatitis C 1 a T RC 20
Hepatitis C 2b T RC 21
Hepatitis C 1 b/2k T RC 22
Hepatitis C 1 c T RC 23
Hepatitis C 2a T RC 24
Hepatitis C 2b T RC 25
Hepatitis C. 2C T RC 26
Hepatitis C 2K T RC 27
Hepatitis C 3a T RC 28
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Hepatitis C 3B T RC 29
Hepatitis C 3k T RC 30
Hepatitis C 4a T RC 31
Hepatitis C 5a T RC 32
Hepatitis C 6a T RC 33
Hepatitis C 6b T RC 34
Hepatitis C 6g T RC 35
Hepatitis C 6d T RC 39
Hepatitis C 6h T RC 37
Hepatitis C 6k T RC 38
Hepatitis C 1 a U 39
Hepatitis C 2b U 40
Hepatitis C 1 b/2k U 41
Hepatitis C 1 c U 42
Hepatitis C 2a U 43
Hepatitis C 2b U 44
Hepatitis C 2C U 45
Hepatitis C 2K U 46
Hepatitis C 3a U 47
Hepatitis C 3B U 48
Hepatitis C 3k U 49
Hepatitis C 4a U 50
Hepatitis C 5a U 51
Hepatitis C 6a U 52
Hepatitis C 6b U 53
Hepatitis C 6g U 54
Hepatitis C 6d U 55
Hepatitis C 6h U 56
Hepatitis C 6k U 57
Hepatitis C 1 a U RC 58
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Hepatitis C 2b U RC 59
Hepatitis C 1 b/2k U RC 60
Hepatitis C 1 c U RC 61
Hepatitis C 2a U RC 62
Hepatitis C 2b U RC 63
Hepatitis C 2C U RC 64
Hepatitis C 2K U RC 65
Hepatitis C 3a U RC 66
Hepatitis C 3B U RC 67
Hepatitis C 3k U RC 68
Hepatitis C 4a U RC 69
Hepatitis C 5a U RC 70
Hepatitis C 6a U RC 71
Hepatitis C 6b U RC 72
Hepatitis C 6g U RC 73
Hepatitis C 6d U RC 74
Hepatitis C 6h U RC 75
Hepatitis C 6k U RC 76
Hepatitis C 1 a genome T 77
Hepatitis C 1a genome UC 78
52 Hepatitis Hepatitis A 1
Hepatitis B 2
Hepatitis D 3
Hepatitis E 4
Hepatitis GA 5
Hepatitis GB 6
Hepatitis A RC 7
Hepatitis B RC 8
Hepatitis D RC 9
Hepatitis E RC 10
Hepatitis GA RC 11
Hepatitis GB RC 12
Hepatitis A U 13
Hepatitis B U 14
CA 02654010 2008-12-01
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Hepatitis D U 15
Hepatitis E U 16
Hepatitis GA U 17
Hepatitis GB U 18
Hepatitis A U RC 19
Hepatitis B U RC 20
Hepatitis D U RC 21
Hepatitis E U RC 22
Hepatitis GA U RC 23
Hepatitis GB U RC 24
53 Human HIV-1 Gp M Clade Al 1
Immunodeficiency HIV-1 Gp M Clade A2 2
virus (HIV) HIV-1 Gp M Clade B 3
HIV-1 Gp M Clade C. 4
HIV-1 Gp M Clade D 5
HIV-1 Gp M Clade Fl 6
HIV-1 Gp M Clade F2 7
HIV-1 Gp M Clade G 8
HIV-1 Gp M Clade H 9
HIV-1 Gp M Clade J 10
HIV-1 Gp M Clade K 11
HIV-1 Gp M CRF A/E 12
HIV-1 Gp M A/G 13
HIV-1 Gp M A/B 14
HIV-1 Gp M A/G/I 15
HIV-1 Gp M F/D 16
HIV-1 Gp M A/G/J 17
HIV-1 Gp M C/B 18
HIV-1 Gp M B/C 19
HIV-1 Gp M A/F/G 20
HIV-1 Gp M C/D 21
HIV-1 Gp M A/E/J/G 22
HIV-1 Gp M B/F 23
HIV-1 Gp M A, CRF01AE 24
HIV-1 Gp M B/G 25
HIV-1 Gp M CRF01AE/B 26
HIV-1 Gp M A2/D 27
HIV-1 Gp M A,F,G,H,J 28
HIV-1 Gp M A/D/G 29
HIV-1 Gp N 30
HIV-1 Gp 0 31
HIV-2 32
HIV-1 Gp M Clade Al RC 33
HIV-1 Gp M Clade A2 RC 34
HIV-1 Gp M Clade B RC 35
HIV-1 Gp M Clade C RC 36
HIV-1 Gp M Clade D RC 37
HIV-1 Gp M Clade Fl RC 38
HIV-1 Gp M Clade F2 RC 39
HIV-1 Gp M Clade G RC 40
HIV-1 Gp M Clade H RC 41
HIV-1 Gp Clade J RC 42
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
HIV-1 Gp M Clade K RC 43
HIV-1 Gp M CRF A/E RC 44
HIV-1 Gp M A/G RC 45
HIV-1 Gp M A/B RC 46
HIV-1 Gp M A/G/I RC 47
HIV-1 Gp M F/D RC 48
HIV-1 Gp M A/G/J RC 49
HIV-1 Gp M C/B RC 50
HIV-1 Gp M B/C RC 51
HIV-1 Gp M A/F/G RC 52
HIV-1 Gp M C/D RC 53
HIV-1 Gp M A/E/J/G RC 54
HIV-1 Gp M B/F RC 55
HIV-1 Gp M A, CRF01AE RC 56
HIV-1 Gp M B/G RC 57
HIV-1 Gp M CRF01AE/B RC 58
HIV-1 Gp M A2/D RC 59
HIV-1 Gp M A,F,G,H,J RC 60
HIV-1 Gp M A/D/G RC 61
HIV-1 Gp N RC. 62
HIV-1 Gp O RC 63
H IV-2 RC 64
HIV-1 Gp M Clade Al U 65
HIV-1 Gp M Clade A2 U 66
HIV-1 Gp M Clade B U 67
HIV-1 Gp M Clade C U 68
HIV-1 Gp M Clade D U 69
HIV-1 Gp M Clade Fl U 70
HIV-1 Gp M Clade F2 U 71
HIV-1 Gp M Clade G U 72
HIV-1 Gp M Clade H U 73
HIV-1 Gp M Clade J U 74
HIV-1 GpMCIadeKU 75
HIV-1 Gp M CRF A/E U 76
HIV-1 GpMA/GU 77
HIV-1 GpMA/BU 78
HIV-1 Gp M A/G/I U 79
HIV-1 Gp M F/D U 80
HIV-1 Gp M A/G/J U 81
HIV-1 Gp M C/B U 82
HIV-1 Gp M B/C U 83
HIV-1 Gp M A/F/G U 84
HIV-1 'Gp M C/D U 85
HIV-1 Gp M A/E/J/G U 86
HIV-1 Gp M B/F U 87
HIV-1 Gp M A, CRF01AE U 88
HIV-1GpMB/GU 89
HIV-1 Gp M CRFOIAE/B U 90
HIV-1 Gp M A2/D U 91
HIV-1 Gp M A,F,G,H,J U 92
HIV-1 Gp M A/D/G U 93
HIV-1 Gp N U 94
HIV-1 Gp O U 95
H IV-2 U 96
HIV-1 Gp M Clade Al U RC 97
HIV-1 Gp M Clade A2 U RC 98
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
HIV-1 Gp M Clade B U RC 99
HIV-1 Gp M Clade C U RC 100
HIV-1 Gp M Clade D U RC 101
HIV-1 Gp M Clade Fl U RC 102
HIV-1 Gp M Clade F2 U RC 103
HIV-1 Gp M Clade G U RC 104
HIV-1 Gp M Clade H U RC 105
HIV-1 Gp M Clade J U RC 106
HIV-1 Gp M Clade K U RC 107
HIV-1 Gp M CRF A/E U RC 108
HIV-1 Gp M A/G U RC 109
HIV-1 Gp M A/B U RC 110
HIV-1 Gp M A/G/I U RC 111
HIV-1 Gp M F/D U RC 112
HIV-1 GpMA/G/JURC 113
HIV-1 GpMC/BURC 114
HIV-1 Gp M B/C U RC 115
HIV-1 Gp M A/F/G U RC 116
HIV-1 Gp M C/D U RC 117
HIV-1 Gp M A/E/J/G U RC 118
HIV-1 Gp M B/F U RC 119
HIV-1 Gp M A,CRF01AE U RC 120
HIV-1 Gp M B/G U RC 121
HIV-1 Gp M CRF01AE/B U RC 122
HIV-1 Gp M A2/D U RC 123
HIV-1 Gp M A,F,G,H,J U RC 124
HIV-1 Gp M A/D/G U RC 125
HIV-1GpNURC 126
HIV-1 GpOURC 127
HIV-2 U RC 128
54 Influenza virus Flu A segment 1 1
Flu A segment 2 2
Flu A segment 3 3
Flu A segment 4 4
Flu A segment 5 5
Flu A segment 6 6
Flu A segment 7 7
Flu A segment 8 8
FIuBHA 9
FIuBNP 10
FIu B PB1 =T 11
FIuBPB2T 12
FIuBNBBAT 13
Flu B M1 BM2 T 14
FIu B PA T 15
Flu B NS1 NS2 T 16
Flu B HA H1N1.T 17
Flu B HA H2N2 T 18
Flu B HA H3N2 T 19
Flu B HA H4N2 T 20
Flu B HA H5N1 T 21
Flu B HA H6N2 T 22
FIuBHAH7N1T 23
Flu B HA H8N4 T 24
Flu B HA H9N2 T 25
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Flu B HA H10N7 T 26
FIuBHAH11N2T 27
FIuBHAH12N1T 28
Flu B HA H13N2 T 29
Flu B HA H14 T . 30
Flu B HA H15N2 T 31
Flu B HA H16N3 T 32
FIuBNAH1N1T 33
FIuBNAH5N1T 34
Flu B NA H3N2 T 35
Flu B NA H3N3 T 36
Flu B NA H8N4 T 37
Flu B NA H6N5 T 38
Flu B NA H4N6 T 39
Flu B NA H7N7 T 40
Flu B NA H3N8 T 41
Flu B NA H2N9 T 42
Flu A segment 1 T RC 43
Flu A segment 2 T RC 44
Flu A segment 3 T RC 45
Flu A segment 4 T RC 46
Flu A segment 5 T RC 47
Flu A segment 6 T RC 48
Flu A segment 7 T RC 49
Flu A segment 8 T RC 50
Flu B HA T RC 51
Flu B NP T RC 52
FIuBPB1TRC 53
Flu B PB2 T RC 54
Flu B NB BA T RC 55
Flu B M1 BM2 T RC 56
FIuBPATRC 57
Flu B NS1 NS2 T RC 58
Flu B HA H1 N1 T RC 59
FIuBHAH2N2TRC 60
FIuBHAH3N2TRC 61
Flu B HA H4N2 T RC 62
Flu B HA H5N1 T RC 63
Flu B HA H6N2 T RC 64
Flu B HA H7N1 T RC 65
Flu B HA H8N4 T RC 66
Flu B HA H9N2 T RC 67
Flu B HA HION7 T RC 68
FIuBHAH11N2TRC. 69
FIuBHAH12N1 TRC 70
FIuBHAH13N2TRC 71
FIuBHAH14TRC 72
Flu B HA H15N2 T RC 73
Flu B HA H16N3 T RC 74
FIuBNAH1N1 TRC 75
Flu. B NA H5N1 T RC 76
Flu B NA H3N2 T RC 77
Flu B NA H8N4 T RC 78
FIuBNAH6N5TRC 79
Flu B' NA H4N6 T RC 80
FIuBNAH7N7TRC 81
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
FIuBNAH3N8TRC 82
Flu B NA H2N9 T RC 83
Flu A segment 1 U 84
Fiu A segment 2 U 85
Flu A segment 3 U 86
Flu A segment 4 U 87
Flu A segment 5 U 88
Flu A segment 6 U 89
Flu A segment 7 U 90
Flu A segment 8 U 91
FIuBHAU 92
FIu B NP U 93
FIuBPB1 U 94
FIu B PB2 U 95
FIuBNBNAU 96
FIuBM1 BM2U 97
FIu B PA U 98
FIuBNS1 NS2U 99
FIu B HA H1N1 U 100
Flu B HA H2N2 U 101
Flu B HA H3N2 U 102
Flu B HA H4N2 U 103
FIuBHAH5N1 U 104
Flu B HA H6N2 U 105
FIu B HA H7N1 U 106
Flu B HA H8N4 U 107
Flu B HA H9N2 U 108
FIuBHAH10N7U 109
FIuBHAH11N2U 110
FIu B HA H12N1 U 111
FIuBHAH13N2U 112
FIuBHAH14U 113
FIu B HA H15N2 U 114
FIuBHAH16N3U 115
FIu B NA H1N1 U 116 '
FIuBNAH5N1 U 117
Flu B NA H3N2 U 118
FIuBNAH8N4U 119
FIuBNAH6N5U 120
FIuBNAH4N6U 121
FIu B NA H7N7 U 122
Flu B NA H3N8 U 123
Flu B NA H2N9 U 124
Flu A segment I U RC 125
Flu A segment 2 U RC 126
Flu A segment 3 U RC 127
Flu A segment 4 U RC 128
Flu A segment 5 U RC 129
Flu A segment 6 U RC 130
Flu A segment 7 U RC 131
Flu A segment 8 U RC 132
FIuBHAURC 133
FIuBNPURC 134
Flu B PB1 U RC 135
FIuBPB2URC 136
FIu B NB BA U RC 137
CA 02654010 2008-12-01
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
FIuBM1 BM2URC' 138
Flu B PA U RC 139
Flu B NS1 N S2 U RC 140
Flu B HA H2N2 U RC 141
Flu B HA H3N2 U RC 142
Flu B HA H4N2 U RC 143
FIuBHAH5N1 URC 144
Flu B HA H6N2 U RC 145
Flu B HA H7N1 U RC 146
Flu B HA H8N4 U RC 147
Flu B HA H9N2 U RC 148
Flu B HA H10N7 U RC 149
FIuBHAH11N2URC 150
Flu B HA H12N1 U RC 151
FIuBHAH13N2URC 152
FIuBHAH14URC 153
Flu B HA H15N2 U RC 154
FIuBHAH16N3URC 155
FIuBNAH1N1 URC 156
FIuBNAH5N1 URC 157
Flu B NA H3N2 U RC 158
Flu B NA H6N5 U RC 159
FIuBNAH7N7URC 160
FIuBNAH3N8URC 161
Flu B NA H2N2 U RC ') 162
55 BK BK virus U 1
BK virus T 2
BfC virus U RC 3
BK virus T RC 4
56 Barmah Barmah Forest virus U 1
Barmah Forest virus T 2
Barmah Forest virus U RC 3
Barmah Forest virus T RC 4
57 Calicivirus Calicivirus U I
Calicivirus T 2
Calicivirus U RC 3
Calicivirus T RC 4
58 Colorado tick fever Colorado tick fever segment 1 U 1
virus Colorado tick fever segment 1 T 2
Colorado tick fever segment 1 U RC 3
Colorado tick fever segment 1 T RC 4
Colorado tick fever segment 2 U 5
Colorado tick fever segment 2 T 6
Colorado tick fever segment 2 U RC 7
Colorado tick fever segment 2 T RC 8
Colorado tick fever se ment 3 U 9
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Colorado tick fever segment 3 T 10
Colorado tick fever segment 3 U RC 11
Colorado tick fever segment 3 T RC 12
Colorado tick fever segment 4 U 13
Colorado tick fever segment 4 T 14
Colorado tick fever segment 4 U RC 15
Coiorado tick fever segment 4 T RC 16
Colorado tick fever segment 5 U 17
Colorado tick fever segment 5 T 18
Colorado tick fever segment 5 U RC 19
Colorado tick fever segment 5 T RC 20
Colorado tick fever segment 6 U 21
Colorado tick fever segment 6 T 22
Colorado tick fever segment 6 U RC 23
Colorado tick fever segment 6 T RC 24
Colorado tick fever segment 7 U 25
Colorado tick fever segment 7 T 26
Colorado tick fever segment 7 U RC 27
Colorado tick fever segment 7 T RC 28
Colorado tick fever segment 8 U 29
Colorado tick fever segment 8 T 30
Colorado tick fever segment 8 U RC 31
Colorado tick fever segment 8 T RC 32
Colorado tick fever segment 9 U 33
Colorado tick fever segment 9 T 34
Colorado tick fever segment 9 U RC 35
Colorado tick fever segment 9 T RC 36
Colorado tick fever segment 10 U 37
Colorado tick fever segment 10 T 38
Colorado tick fever segment 10 U RC 39
Colorado tick fever segment 10 T RC 40
Colorado tick fever segment 11 U 41
Colorado tick fever segment 11 T 42
Colorado tick fever segment 11 U RC 43
Coloradd tick fever segment 11 T RC 44
Colorado tick fever segment 12 U 45
Colorado tick fever segment 12 T 46
Colorado tick fever segment 12 U RC 47
Colorado tick fever segment 12 T RC 48
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
59 Foot and Mouth Foot and Mouth virus A U 1
Disease Foot and Mouth virus A T 2
Foot and Mouth virus A U RC 3
Foot and Mouth virus A T RC 4
Foot and Mouth virus Asia 1 U 5
Foot and Mouth virus Asia 1 T 6
Foot and Mouth virus Asia 1 U RC 7
Foot and Mouth virus Asia 1 T RC 8
Foot and Mouth virus C U 9
Foot and Mouth virus C T 10
Foot and Mouth virus C U RC 11
Foot and Mouth virus C T RC 12
Foot and Mouth virus 0 U 13
Foot and Mouth virus 0 T 14
Foot and Mouth virus 0 U RC 15
Foot and Mouth virus 0 T RC 16
Foot and Mouth virus SAT I U 17
Foot and Mouth virus SAT 1 T 18
Foot and Mouth virus SAT 1 U.RC 19
Foot and Mouth virus SAT 1 T RC 20
Foot and Mouth virus SAT 2 U 21
Foot and Mouth virus SAT 2 T 22
Foot and Mouth virus SAT 2 U RC 23
Foot and Mouth virus SAT 2 T RC 24
Foot and Mouth virus SAT 3 U 25
Foot and Mouth virus SAT 3 T 26
Foot and Mouth virus SAT 3 U RC 27
Foot and Mouth virus SAT 3 T RC 28
60 Hepatitis GB virus GB virus C U 1
GB virus C T 2
GBvirusCURC 3
GB virus C T RC 4
61 Hendra virus Hendra virus U 1
Hendra virus T 2
Hendra virus U RC 3
Hendra virus T RC 4
62 Human adenovirus Human adenovirus A U 1
Human adenovirus A T 2
Human adenovirus A U RC 3
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Human adenovirus A T RC 4
Human adenovirus B U 5
Human adenovirus B T 6
Human adenovirus B U RC 7
Human adenovirus B T RC 8
Human adenovirus C U 9
Human'adenovirus C T 10
Human adenovirus C RC 11
Human adenovirus C T RC 12
Human adenovirus D U 13
Human adenovirus D T 14
Human adenovirus D U RC 15
Human adenovirus D T RC 16
Human adenovirus E U 17
Human adenovirus E T 18
Human adenovirus E U RC 19
Human adenovirus E T RC 20
Human adenovirus F U 21
Human adenovirus F T 22
Human adenovirus F U RC 23
Human adenovirus F T RC 24
63 Human astrovirus Human astrovirus U 1
Human astrovirus T 2
Human astrovirus U RC 3
Human astrovirus T RC 4
64 Human bocavirus Human bocavirus U I
Human bocavirus T 2
Human bocavirus U RC 3
Human bocavirus T RC 4
65 Human coronavirus Human coronavirus 229E U I
Human coronavirus 229E T 2
Human coronavirus 229E U RC 3
Human coronavirus 229E T RC 4
Human coronavirus.HKU1 type A U 5
Human coronavirus HKU1 type A T 6
Human coronavirus HKU1 type A U RC 7
Human coronavirus HKUI type A T RC 8
Human coronavirus NL63 U 9
Human coronavirus NL63 T 10
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Human coronavirus NL63 U RC 11
Human coronavirus NL63 T RC 12
Human coronavirus OU43 U 13
Human coronavirus OU43 T 14
Human coronavirus OU43 U RC 15
Human coronavirus OU43 T RC 16
66 Human enterovirus Human enterovirus-A U 1
Human enterovirus-A T 2
Human enterovirus A-U RC 3
Human enterovirus A-T RC 4
Human enterovirus-B U 5
Human enterovirus-B T 6
Human enterovirus-B U RC 7
Human enterovirus-B T RC 8
Human enterovirus-C U 9
Human enterovirus-C T 10
Human enterovirus-C U RC 11
Human enterovirus-C T RC 12
Human enterovirus-D U 13
Human enterovirus-D T 14
Human enterovirus-D U RC 15
Human enterovirus-D T RC 16
67 Human Herpes Virus Human Herpes Virus-I U 1
Human Herpes Virus-I T 2
Human Herpes Virus-1 U RC 3
Human Herpes Virus-1 T RC 4
Human Herpes Virus-2 U 5
Human Herpes Virus-2 T 6
Human Herpes Virus-2 U RC 7
Human Herpes Virus-2 T RC 8
Human Herpes Virus-3 U 9
Human Herpes Virus-3 T 10
Human Herpes Virus-3 U RC 11
Human Herpes Virus-3 T RC 12
Human Herpes Virus-4 U 13
Human Herpes Virus-4 T 14
Human Herpes Virus-4 U RC 15
Human Herpes Virus-4 T RC 16
Human Herpes Virus-5 AD169 U 17
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Human Herpes Virus-5 (AD169) T 18
Human Herpes Virus-5 (AD169) U RC 19
Human Herpes Virus-5 T(AD169) RC 20
Human Herpes Virus-6A U 21
Human Herpes Virus-6A T 22
Human Herpes Virus-6A URC 23
Human Herpes Virus-6A T RC 24
Human Herpes Virus-6B U 25
Human Herpes Virus-6B T 26
Human Herpes Virus-6B URC 27
Human Herpes Virus-6B T RC 28
Human Herpes Virus-7 U 29
Human Herpes Virus-7 T 30
Human Herpes Virus-7 U RC 31
Human Herpes Virus-7 T RC 32
Human Herpes Virus-8 U 33
Human Herpes Virus-8 T 34
Human Herpes Virus-8 U RC 35
Human Herpes Virus-8 T RC 36
68 Human Human metapneumovirus U 1
metapneumovirus Human metapneumovirus T 2
Human metapneumovirus U RC 3
Human metapneumovirus T RC 4
69 Human Human Parainfluenza-1 U 1
Parainfluenza Human Parainfluenza-1 T 2
Human Parainfiuenza-1 U RC 3
Human Parainfluenza-1 T RC 4
Human Parainfluenza-2 U 5
Human Parainfluenza-2 T 6
Human Parainfluenza-2 U RC 7
Human Parainfluenza-2 T RC 8
Human Parainfluenza-3 U 9
Human Parainfluenza-3 T 10
Human Parainfluenza-3 U RC 11
Human Parainfluenza-3 T RC 12
Human parechovirus Human parechovirus U 1
Human parechovirus T 2
Human parechovirus U RC 3
Human parechovirus T RC 4
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
71 Human Rhinovirus Human Rhinovirus-A U 1
Human Rhinovirus-A T 2
Human Rhinovirus-A U RC 3
Human Rhinovirus-A T RC 4
Human Rhinovirus-B U ' 5
Human Rhinovirus-B T ~ 6
Human Rhinovirus-B U RC 7
Human Rhinovirus-B T RC 8
72 Human respiratory Human RSV U I
syncytial virus Human RSV T 2
HumanRSVURC 3
Human RSV T RC 4
73 Measles virus Measles virus U 1
Measles virus T 2
Measles virus RC U 3
Measles virus RC T 4
74 Mumps virus Mumps virus U 1
Mumps virus T 2
Mumps virus RC U 3
Mumps virus RC T 4
75 Norovirus Norovirus U 1
Norovirus T 2
Norovirus RC U 3
Norovirus RC T 4
76 Norwalk virus Norwalk virus U I
Norwalk virus T 2
Norwalk virus RC U 3
Norwalk virus RC T 4
77 Parvovirus B19 Parvovirus B19 U 1
Parvovirus B19 T 2
Parvovirus B19 RC U 3
Parvovirus B19 RC T 4
78 Poliovirus. Poliovirus U 1
Poliovirus T 2
Poliovirus RC U 3
Poliovirus RC T 4
79 Rabies virus Rabies virus U 1
Rabies virus T 2
Rabies virus RC U 3
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Rabies virus RC T 4
80 Ross River virus Ross River virus U I
Ross River virus T 2
Ross River virus RC U 3
Ross River virus RC T 4
81 Rotavirus Rotavirus A NSPI U I
Rotavirus A NSPI T 2
Rotavirus A NSP1 U RC 3
Rotavirus A NSP1 T RC 4
Rotavirus B NSP1 U 5
Rotavirus B NSP1 T ' 6
Rotavirus B NSP1 U RC 7
Rotavirus B NSP1 T RC 8
Rotavirus C NSP1 U 9
Rotavirus C NSP1 T 10
Rotavirus C NSP1 U RC 11
Rotavirus C NSP1 T RC 12
Rotavirus A NSP2 U 13
Rotavirus A NSP2 T 14
Rotavirus A NSP2 U RC 15
Rotavirus A NSP2 T RC 16
Rotavirus B NSP2 U 17
Rotavirus B NSP2 T 18
Rotavirus B NSP2 U RC 19
Rotavirus B NSP2 T RC 20
Rotavirus C NSP2 U 21
Rotavirus C NSP2 T 22
Rotavirus C NSP2 U RC 23
Rotavirus C NSP2 T RC 24
Rotavirus A NSP3 U 25
Rotavirus A NSP3 T 26
Rotavirus A NSP3 U RC 27
Rotavirus.A NSP3 T RC 28
Rotavirus B NSP3 U 29
Rotavirus B NSP3 T 30
Rotavirus B NSP3 U RC 31
Rotavirus B NSP3 T RC 32
Rotavirus C NSP3 U 33
Rotavirus C NSP3 T 34
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Sequence Microorganism' Sequence SEQ ID
Listing # NO:
Rotavirus C NSP3 U RC 35
Rotavirus C NSP3 T RC 36
Rotavirus A NSP4 U 37
Rotavirus A NSP4 T 38
Rotavirus A NSP4 U RCG- 39
Rotavirus A NSP4 T RC 40
Rotavirus B NSP4-U 41
Rotavirus B NSP4 T 42
Rotavirus B NSP4 U RC 43
Rotavirus B NSP4 T RC 44
Rotavirus C NSP4 U 45
Rotavirus C NSP4 T 46
Rotavirus C NSP4 U RC 47
Rotavirus C NSP4 T RC 48
Rotavirus A NSP5 U 49
Rotavirus A NSP5 T 50
Rotavirus A NSP5 U RC 51
Rotavirus A NSP5 T RC 52
Rotavirus B NSP5 U 53
Rotavirus B NSP5 T 54
Rotavirus B NSP5 U RC 55
Rotavirus B NSP5 T RC 56
Rotavirus C NSP5 U 57
Rotavirus C NSP5 T 58
Rotavirus C NSP5 U RC 59
Rotavirus C NSP5 T RC 60
Rotavirus A VP1 U 61
Rotavirus A VPI T 62
Rotavirus A VP1 U RC 63
Rotavirus A VPI T RC 64
Rotavirus C VP1 U 65
Rotavirus C VP1 T 66
Rotavirus C VPI U RC 67
Rotavirus C VPI T RC 68
Rotavirus A VP2 U 69
Rotavirus A VP2 T 70
Rotavirus A VP2 U RC 71
Rotavirus A VP2 T RC 72
Rotavirus B VP2 U 73
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Rotavirus B VP2 T 74
Rotavirus B VP2 U RC 75
Rotavirus B VP2 T RC 76
Rotavirus C VP2 U 77
Rotavirus C VP2 T 78
Rotavirus C VP2 U RC 79
Rotavirus C VP2 T RC 80
Rotavirus A VP3 U 81
Rotavirus A VP3 T 82
Rotavirus A VP3 U RC 83
Rotavirus A VP3 T RC 84
Rotavirus C VP3 U 85
Rotavirus C VP3 T 86
Rotavirus C VP3 U RC 87
Rotavirus C VP3 T RC 88
Rotavirus A VP4 U 89
Rotavirus A VP4 T 90
Rotavirus A VP4 U RC 91
Rotavirus A VP4 T RC 92
Rotavirus B VP4 U 93
Rotavirus B VP4 T 94
Rotavirus B VP4 U RC 95
Rotavirus B VP4 T RC 96
Rotavirus C VP4 U 97
Rotavirus C VP4 T 98
Rotavirus C VP4 U RC 99
Rotavirus C VP4 T RC 100
Rotavirus A VP6 U 101
Rotavirus A VP6 T 102
Rotavirus A VP6 U RC 103
Rotavirus A VP6 T RC 104
Rotavirus B VP6 U 105
Rotavirus B VP6 T 106
Rotavirus B VP6 U RC 107
Rotavirus B VP6 T RC 108
Rotavirus C VP6 U 109
Rotavirus C VP6 T 110
Rotavirus C VP6 U RC 111
Rotavirus C VP6 T RC 112
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Sequence Microorganism Sequence SEQ ID
Listin # NO:
Rotavirus A VP7 U 113
Rotavirus A VP7 T 114
Rotavirus A VP7 U RC 115
Rotavirus A VP7 T RC 116
Rotavirus B VP7 U 117
Rotavirus B VP7 T 118
Rotavirus B VP7 U RC 119
Rotavirus B VP7 T RC 120
Rotavirus C VP7 U 121
Rotavirus C VP7 T 122
Rotavirus C VP7 U RC 123
Rotavirus C VP7 T RC 124
82 SARS coronavirus SARS coronavirus U 1
SARS coronavirus T 2
SARS coronavirus RC U 3
SARS coronavirus RC T 4
83 TT virus TT virus U 1
TT virus T 2
TT virus RC U 3
TT virus RC T 4
84 TTV minivirus TTV minivirus U 1
TTV minivirus T 2
TTV minivirus U RC 3
TTV minivirus T RC 4
85 West Nile virus West Nile virus U 1
West Nile virus T 2
West Nile virus U RC 3
West Nile virus T RC 4
86 Alpha Virus Alpha Virus U 1
Alpha Virus T 2
Alpha Virus U RC 3
AI ha Virus T RC 4
87 Camel pox Camel pox U I
Camel pox T 2
Camel pox U RC 3
Camel po T RC 4
88 Cow pox Cow pox U 1
Cow pox T 2
Cow pox U RC 3
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Cow ox T RC 4
89 Coxiella burnetii Coxiella burnetii U 1
Coxiella burnetii T 2
Coxielia burnetii U RC 3
Coxiella burnetii T RC 4
90 Crimean-Congo HF Crimean-Congo HF S U I
Crimean-Congo HF S T 2
Crimean-Congo HF S U RC 3
Crimean-Congo HF S T RC 4
Crimean-Congo HF L U 5
Crimean-Congo HF L T 6
Crimean-Congo HF L U RC 7
Crimean-Congo HF L T RC 8
Crimean-Congo HF M U 9
Crimean-Congo HF M T 10
Crimean-Congo HF M U RC 11
Crimean-Congo HF M T RC 12
91 Dengue virus Dengue virus-1 U 1
Dengue virus-1 T 2
Dengue virus-1 U RC 3
Dengue virus-1 T RC 4
Dengue virus-2 U 5
Dengue virus-2 T 6
Dengue virus-2 U RC 7
Dengue virus-2 T RC 8
Dengue virus-3 U 9
Dengue virus-3 T 10
Dengue virus-3 U RC 11
Dengue virus-3 T RC 12
Dengue virus-4 U 13
Dengue virus-4 T 14
Dengue virus-4 U RC 15
Dengue virus-4 T RC 16
92 Eastern Equine Eastern Equine Encephalitis U 1
Encephalitis Eastern Equine Encephalitis T 2
Eastern Equine Encephalitis U RC 3
Eastern Equine Encephalitis T RC 4
93 Ebola virus Ebola Sudan U I
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Ebola Sudan T 2
Ebola Sudan U RC 3
Ebola Sudan T RC 4
Ebola Reston U 5
Ebola Reston T 6
Ebola Reston U RC 7
Ebola Reston T RC 8
Ebola Zaire U 9
Ebola Zaire T 10
Ebola Zaire U RC 11
Ebola Zaire T RC 12
94 Marburg virus Marburg virus U I
Marburg virus T 2
Marburg virus U RC 3
Marburg virus T RC 4
-95 Guanarito virus Guanarito virus L U I
Guanarito virus L T 2
Guanarito virus L U RC 3
Guanarito virus L T RC 4
Guanarito virus S U 5
Guanarito virus S T 6
Guanarito virus S U RC 7
Guanarito virus S T RC 8
96 Hanta virus Hanta virus M U 1
Hanta virus M T 2
Hanta virus M U RC 3
Hanta virus M T RC 4
Hanta virus L U 5
Hanta virus L T 6
Hanta virus L U RC 7
Hanta virus L T RC 8
Hanta virus S'U 9
Hanta virus S T 10
Hanta virus S U RC 11
Hanta virus S T RC 12
97 Hantaan virus Hantaan virus L U 1
Hantaan virus L T 2
Hantaan virus L U RC 3
Hantaan virus L T RC 4
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Hantaan virus M U 5
Hantaan virus M T 6
Hantaan virus L M RC 7
Hantaan virus L M RC 8
Hantaan virus S U 9
Hantaan virus S T 10
Hantaan virus S U RC 11
Hantaan virus S T RC 12
98 Japanese Japanese encephalitis virus U I
encephalitis virus Japanese encephalitis virus T 2
Japanese encephalitis virus U RC 3
Japanese encephalitis virus T RC 4
99 Junin virus Junin virus S U 1
Junin virus S T 2
Junin virus S U RC 3
Junin virus S T RC 4
Junin virus L U 5
Junin virus L T 6
Junin virus L U RC 7
Junin virus L T RC 8
100 Lassa virus Lassa virus L U I
Lassa virus L T 2
Lassa virus L U RC 3
Lassa virus L T RC 4
Lassa virus S U 5
Lassa virus S T 6
Lassa virus S U RC 7
Lassa virus S T RC 8
101 Machupo virus Machupo virus L U I
Machupo virus L T 2
Machupo virus L U RC 3
Machupo virus L T RC 4
Machupo virus S U 5
Machupo virus S T 6
Machupo virus S U RC 7
Machupo virus S T RC 8
102 Monkeypox virus Monkeypox virus U 1
Monke ox virus T 2
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Sequence Microorganism Sequence SEQ ID
Listing # NO:
Monkeypox virus U RC 3
Monkeypox virus T RC 4
103 Murray Valley Murray Valley encephalitis U 1
encephalitis Murray Valley encephalitis T 2
Murray Valley encephalitis U RC 3
Murray Valley encephalitis T RC 4
104 Nipah virus Nipah,virus U 1
Nipah virus T 2
Nipah virus U RC 3
Nipah virus T RC 4
105 Rift Valley Fever Rift Valley Fever virus S U 1
virus Rift Valley Fever virus S T 2
Rift Valley Fever virus S U RC 3
Rift Valley Fever virus S T RC 4
Rift Valley Fever virus M U 5
Rift Valley Fever virus M T 6
Rift Valley Fever virus M U RC 7
Rift Valley Fever virus M- T RC 8
Rift Valley Fever virus L U 9
Rift Valley Fever virus L T 10
Rift Valley Fever virus L U RC 11
Rift Valle Fever virus L T RC 12
106 Sabia virus Sabia virus S U 1
Sabia virus S T 2
Sabia virus S U RC 3
Sabia virus S T RC 4
Sabia virus L U 5
Sabia virus L T 6
Sabia virus L U RC 7
Sabia virus L T RC 8
107 Sin Nombre virus Sin Nombre virus L U 1
Sin Nombre virus L T 2
Sin Nombre virus L U RC 3
Sin Nombre virus L T RC 4
Sin.Nombre virus S U 5
Sin Nombre virus S T 6
Sin Nombre virus S U RC 7
Sin Nombre virus S T RC 8
Sin Nombre virus M U 9
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Sequenae Microorganism Sequence SEQ ID
Listing # NO:
Sin Nombre virus M T 10
Sin Nombre virus M U RC 11
Sin Nombre virus M T RC 12
108 Variola major Variola major U 1
Variola major T 2
Variola major U RC 3
Variola major T RC 4
109 Variola minor Variola minor U 1
Variola minor T 2
Variola minor U RC 3
Variola minor T RC 4
110 Venezuelan equine Venezuelan equine encephalitis U 1
encephalitis Venezuelan equine encephalitis T 2
Venezuelan equine encephalitis U RC 3
Venezuelan equine encephalitis T RC 4
111 Western equine Western equine encephalitis U 1
encephalitis Western equine encephalitis T 2
Western equine encephalitis U RC 3
Western equine encephalitis l` RC 4
112 Yellow Fever virus Yellow Fever virus U 1
Yellow Fever virus T 2
Yellow Fever virus U RC 3
Yellow Fever virus T RC 4
*U derivative top strand with all cytosine replaced with uracil
*T modified top strand with all uracil replaced with thymine
*U RC derivative bottom strand with all cytosine replaced with uracil
*T RC modified bottom strand with all uracil replaced with thymine
5
It will be appreciated by persons skilled in the art that numerous variations
and/or
modificatioris may be made to the invention as shown in the specific
embodiments
without departing from the spirit or scope of the 'invention as broadly
described. The
present embodiments are, therefore, to be considered in all respects as
illustrative and
10 not restrictive.
