Note: Descriptions are shown in the official language in which they were submitted.
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WO92/112~3 PCT/US91/09688
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PCR PRIMERS FOR DETECTION OF LEGIONELLA SPECIES AND METHODS FOR
CONTROL'IN6 VISUAL INTENSITY IN HYBRIDIZATION ASSAYS
BACKGROUND OF THE INVENTION
1. Field of the Invention.
This in~ention provides for superior nucleic acid
primers for amplification of select target regions of the
genome of the genus Legionella. The invention ~acilitates
detection of pathogenic and nonpathogenic forms of this genus.
The invention further provides for processes for using the
primers in template dependent nucleic acid polymerase extension
reactions to amplify select target regions. Kits for the use -
of these primers are also provided.
This invention further provides for methods of
controlling the intensity of visual signal for detection of
duplex formation in nucleic acid hybridization assays under -
high stringent conditions. This method involves the blending
of different capture probes onto a solid support.
2. Information Disclosure.
Legionella species are known as both pathogenic and
nonpathogenic microorganisms. In the pneumonic form, they are
intracellular pathogens of lung macrophage cells.
Legionellaceae, Chpt. 9260 J. in Standard Methods of the
Examination of Water and Wastewater, Eds. Clesceri, Greenberg
30 and Trussell, 17th Ed. 1989 pages 9-149 to 9-153 and Muraca,
P.W. et al., 1988, Environmental Aspects of Legionnaires'
Disease, J. Amer. Water Works Assoc. 80:78-86.
A surface antigen of Legionella has been implicated
as a requirement for intracellular pathogenicity and is called
a macrophage infecti~ity potentiator or nip. Cianciotto, N.P.
et al., 1989, A Legionella pneumophila Gene Encoding a
Species-specific Surface Protein Potentiates Initiation of
Intracellular Infection, Infection and Immunity, 57:1255-126~.
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WO92/11273 PCT/US91/09688
The nucleotide sequence of the 5S rRNA has been
reported by MacDonnell, M.T. and R.R. Colwell, 1987, The
Nucleotide Sequence of the 5S rRNA From Legionella pneumophila,
Nucleic Acid Research, 15:1335; and, by Chumakov, K.M et al.,
1986, Use of 5S Ribosomal RNA Nucleotide Sequence Analysis for
the Study of Phylogeny of the Genus Legionella, Mol._Genet..
Mikrobiol Vlrusol., 8:38-40.
The nucleotide and amino acid sequence of the
macrophage infectivity protein is known and reported by
Engleberg, N.C. et al., 1989, DNA Sequence of mip, a Legionella
pneumophila Gene Associated With Macrophage Infectivity,
Infection and Immunity, 57:1263-1270.
Standard culture techniques for Legionella are not
adequate to properly assess risks from this deadly pathogen.
Hussong, D. et al, 1987, Viable Legionella pneumophila Not
Detectable by Culture on Agar Media, Bio/Technology 5:947-950. -~
Nucleic acid probes for detection of Legionella ~ :
pneumophila have been reported. Grimont, P.A.D. e~ al., 1985,
DNA Probe Specific for Legionella pneumop~ila, J. Clin. Micro.,
21:431-437; and Engleberg, N.C. et al., 1986, A Legionella-
Specific DNA Probe Detects Organisms in Lung Tissue Homogenates
from Intranasally Inoculated Mice, Israel J. of Med. Sciences,
22:703-705.
The use of polymerase chain reaction amplification
methods to detect Legionella species has been disclosed by
Starnbach, M.N. et al., 1989, Species-Specific Detection of
Legio~ella pneumophila in Water by DNA Amplification and
Hybridization, J. of Clin. Microbiol., 27:1257-1261; in
U.S.S.N. 07/467,813 filed on January 1, 1990; and, also in
Detection of Legionella in Environmental Samples Using
Polymerase Chain Reaction (PCR), Biotechnology Bulletin, May
1990 pages 11-12.
SUMMARY OF THE INVENTION
This invention provides for superior amplification
primers for use in assays for detecting pathogenic Legionella .
species. Described primers are able to amplify select regions
of the 5 S RNA gene that are uniquely common to most species of
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0 92/tl273 PC~r/US91/09688
Legionella. Other primers are able to discriminate between
Legionella having the mlp gene and those without the mip gene.
The primers are further advantageous in that they have similar
thermal melting points and can be used in combination to
efficiently and equally amplify more than one region of the
Legionella genome in a single amplification reaction mixture.
In particular, this invention provides for a
composition comprising multiple pairs of amplification primers
for amplifying subsequences of the 5 S RNA gene and mip gene of
Legionella species each primer having a thermal melting point
with respect to their binding sites of less than about 8C of
each other. Methods for amplifying genomic regions of
~egionella using these compositions are also described herein.
This invention further provides for nucleic acid
polymerase primers for the amplification of subsequences of
nucleic acid from Legionella species wherein the primers bind
substantially to the nucleic acid su~sequence selected from the
group consisting of (a) 3'-CGTA~CCACGGCTAAACC-5'; Seq. ID No.
12; ~b) 3'-CGAAACGGTAGTTTAGA~AGACTT-5'; Seq. ID No. 13; (c) 3'-
CCGCTGATATCGCYAAACCTT-5'; Seq. ID No. 14; (d) 3'-
CGCTACTGGATGAAAGYGTACT-5'; Seq. ID No. 15; (e) 3'-
CCGCTGATATCGCCACACCTT-5'; Seq. ID No. 18; (f) 3'- -
CGCTACTGGATGA~AGCGTAC-5'; Seq. ID No. 19; and (g) 3'-
CA~AAC>AGTTTAGAAAAACTT-5'; Seq. ID No. 20 where Y represents
a cytosine or thymine base and the primers having a base Y
encompass a mixture of the two primers. Examples of such
primers are: (a) 5'-GCATTGGTGCCGATTTGG-3'; Seq. ID No. 6; (b)
5'-GCTTTGCCATCA~ATCTTTCTGAA-3'; Seq. ID No. 7; (c) 5'-
GGCGACTATAGCGRTTTGGAA-3'; Seq. ID No. 10; (d)
5'-GCGATGACCTACTTTCRCATGA-3'; Seq. ID No. 9; (e) 5'-
GGCGACTATAGCGGTGTGGAA-3'; Seq. ID No. 21; (f) 5'-
GCGATGACCTAC m CGCATG-3'; Seq. ID No. 22; and (g) 5'-
GTTTTGCCATCA~ATCTTTTTGAA-3'; Seq. ID No. 23 where R represents
an adenine or a guanlne base and the primers having a base R
are a mixture of the two primers.
This invention also provides for an internal positive
control oligonucleotide sequence comprising an unnatural
oligonucleotide subsequence ~lanked by primer binding sites.
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W092/ll273 ~ 7 ~ ~ V~ 3 PCT/US91/09688
In this fashion the IPC will amplify with the same primer pairs
used to amplify the target subsequence. In one embodiment of
the present invention, the target is the mip gene of Legionella
species. In this embodiment the IPC comprises an unnatural
subsequence flanked by the following nucleic acid subsequences:
(a) 3'-CGTAACCACGGCTAAACC-5'; Seq. ID No. 12; and, (b) 3'-
CGAAACGGTAGTTTAGAAAGACTT-5'; Seq. ID No. 13. In another
embodiment the primer binding sites of the IPC will be
identical to the primer binding sites of the 5 S RNA gene
target. In this embodiment the primer binding sit~s will
comprise
(c) 3'-CCGCTGATATCGCYAAACCTT-5'; Seq. ID No. 14; and/or,
(d) 3'-CGCTACTG&ATGAAAGYGTACT-5'; Seq. ID No. 15; and/or,
(e) 3'-CCGCTGATATCGCCACACCTT-5'; Seq. ID No. 18; and/or,
(f) 3'-CGCTACTGGATGAAAGCGTAC-5'; Seq. ID No. 19 where Y
represents a cytosine or thymine base. More specifically there
is disclosed an IPC oligonucleotide sequence ha~ing either one
or both of the following sequences 5'-GCATTGGTGCCGATTTGG-3';
Seq. ID No. 6 and 5'-TTCAGAAAGATTTGATGGCAAAGC-3'; Seq. ID No.
13.
It should be understood that alternative primers
could be envisioned deviating from those described. Deviations
might include minor base changes, biotinylated bases, base
analogs or additional bases added to the primer ends. Wherever ~ -
such modifications or changes do not substantially affect the
primers' ability to amplify the target region internal to the
stated binding sites for the named primers such modified
primers are considered to be within the scope of this
invention.
This invention further embraces processes utilizing
the above amplification primers. Preferably in a multiplex PCR
format. In particular there is disclosed a process of using
nucleotide polymerases for amplification of nucleic acid
subsequences from Legionella species wherein the polymerases
initiate subsequence amplification by extension of nucleic acid
primers which bind substantially to a nucleic acid subsequence
selected from the group consisting of: (a) 3'-
CGTAACCACGGCTAAACC-5'; Seq. ID No. 12; (b) 3'-
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CGAAACGGTAGTTTAGAAAGACTT-5'; Seq. ID No. 13; (c) 3'-
CCGCTGATATCGCYAAACCTT-5'; Seq. ID No. 14; (d) 3'-
CGCTACTGGATGAAAGYGTACT-5'; Seq. ID No. 15; (e) 3'-
CCGCTGATATCGCCACACCTT-5'; Seq. ID No. 18; (f) 3'-
CGCTACTGGATGAAAGCGTAC-5'; Seq. ID No. 19; and (g) 3'-
CAAAACGGTAGTTTAGAAAAACTT-5'; Seq. ID No. 20 where Y represents
a cytosine or thymine base and the primers having a base Y are
a mixture of the two primers. This invention also provides for
the above process where the primers are as described above and
where an internal positive control oligonucleotide sequence is
included in the amplification mixture.
This invention provides for kits for the
amplification of nucleic acid from Le~ionella species
comprising a compartment which contains a nucleic acid which
binds substantially to a nucleic acid subsequence as described
above and preferably containing the primers as described above.
Internal positive control oligonucleotide sequence are also
included in an alternative embodiment of these kits.
A second aspect of the disclosed invention relates to
controlling the in~ensity of the visual signal used to detect
duplex formation in nucleic acid hybridization assays. The
model is a nucleic acid hybridization assay kit for the
detection of a particular genus having numerous distinct
species. In some cases detection and discrimination of a
particular species may be desirable especially where a
particular species is known to be of medical importance. In
other cases where most of the species of organisms within a
genus have been associated with disease, it is desirable to
detect the presence the entire genus of organisms. It is
further desirable that the detection of the genus of organisms
be obtained as one positive visual response regardless of
species. If the target nucleic acid sequence used for the
detection of the genus of organisms varies for different
species within the genus it is typically necessary to lower the
stringency of hybridization or utilize multiple capture probes.
This invention avoids this problem of resorting to low
stringent conditions.
WO92/ll273 ~~9 3 6 PCT/US91/09688
Lower stringent conditions are routinely used to
accommodate the capture of multiple target se~ences that
contain variations in their nucleic acid sequences. The
stringency is reduced by either lowering the temperature of
hybridization and wash or by modification of the buffer. When
the stringent conditions are reduced and the target nucleic
acid sequence is very similar to nucleic acid sequences of ~ -
another genus of organisms specificity of the capture probe for
the target genus can be lost.
When multiple capture probes are used and are
selected to be compatible to variations in the target nucleic
acid sequences, the specificity under high stringent conditions
can be regained. The blending of multiple probes permits a
single positive response for the presence of a group of target
organisms. Without this invention, the different capture
probes would normally be immobilized individually on a solid -
support. As a result, the assay would require the use of more
test sample, more time to perform the test and more
interpretation by the user.
In those kits utilizing this invention, the multiple
capturP probes are blended together and immobilized in such a
manner to accommodate the detection of the various species of
the genus of organisms in a single test. The probes are
selected to be compatible to blending (i.e. contain
noncomplementary sequence and have as similar as possible
melting temperatures). The capture probes should be blended in
the proper ratio to generate a result of equal intensity for
species within the genus of organisms to minimize
interpretation of the result when semiquantitation and equal
sensitivity for all target organisms are desirable. Depending
on the hybridization characteristics, the concentration each
capture probe in the blended may or may not be equal. It is
possible to determine the proper capture probe blend by a
series of titrations. The blends of the capture probes of
varying ratios are created and immobilized on a solid support.
The capture probes are individually immobilized on
the solid support for reference in the titration of the probe
blend. Nucleic acid from different species within the genus of
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organisms to be tested are hybridized to the capture probe
blends and to the individual probes. The probe blend that
generates the same response as an individual specific probe for
the species within the genus is chosen for use as a capture
probe blend in the kit. These capture probes may also be
individually be immobilized as capture probes should further
speciation be desired but it is not necessary.
It should be noted that the mixture of capture probes
may or may not contain all probes for the same gene provided
that they have similar hybridization characteristics.
The mixture of capture probes may be able to capture
more than one genus if desired. For example, should a kit be
developed that detects food borne pathologies such as the genus
Salmonella and Shigella, a mixture of probes can be used to
indicate a pathogen is present of either of the two genera.
This may be all that is necessary for the food laboratory. If
further identification is necessary (maybe for food poisoning
problem) then the same capture probes may be used for
identification in separate and unblended states.
More specifically, this invention provides for a
method for controlling visual density of detection probes bound
to capture probes in a nucleic acid hybridization assay having
a first and a second capture probe wherein the nucleic acid
sequences of the two capture probes are different, said method
comprising: (a) mixing a blend of the two different capture
probes; (b) affixing the mixture of probes from step (a) and
the first capture probe to discrete first and second regions of
a solid support wherein the concentration of the first capture
probe is equal in both regions; (c) binding the detection
probes to the capture probes of step (b) under high stringent
hybridization conditions such that the detection probes bind to
one capture probe but not the other; and (d) detecting
hybridization of the detection probe by visual density; wherein
the proportion of the two capture probes in the blend of step
(a) i9 adjusted to gi~e a visual density in the first region
equal to that of the second region having the first capture
probe when equal amounts of the detection probes are present in
a test sample. The concentration of the second probe in the
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WO92/11273 PCT/US91/09688
second region will not be the same as the concentration of the
second probe in the first region. Typically the second probe
is not present in the second region.
The detection probe can be an amplification product
such as a PCR amplification product. The assay format can
involve a detection probe which is a complex of a target
nucleic acid which binds under high stringent hybridization
conditions to different regions of the capture and signal probe
and wherein the capture and signal probes do not hybridize to
each other.
Furthermore it is preferred that this method
deliberately provide for a proportion of the two capture probes
adjusted according to a known quantity of target nucleic acid.
The detection range for the assay is in a concentration range
of between about 10 x 10~12 to about 100 x 10-9 molar. It
being desired that the assay provide a positive gradient of
visual density over a target nucleic acid concentration of -~ -
between about 10 x 10-12 to about 100 x 10-9 molar.
Kits embracing the above method are also a part of
this invention. In particular there is contemplated a nucleic
acid hybridization kit providing equal visual density for
positive results in a test sample. The kit uses detection
probes and a first and a second capture probe wherein the
capture probes have different nucleic acid sequences, said kit
comprising the following components: (a) a solid support having
a first discrete region having a concentration of the first and
second capture probes affixed thereto; (b) a solid support
having a second discrete region wherein the first capture
probes are affixed thereto in a concentration equal to the
concentration of the first probe in the mixture described in
component (a); and, (c) a container containing detection
probes; wherein the proportion of the two capture probes in the
mixture of component (a) is adjusted to give a visual density
equal to that of the solid support of component (b) under
identical hybridization conditions and with equal amounts of
the detection probes present in the test sample. The
concentration of second probe in the second region is not the
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092/11273 PCT/US91/09688
same as its concentration in the first region and is typically
not present.
DEFINITIONS
"Affixing" in the context of nucleic acids and solid
supports refers to the binding or attaching of the nucleic
acids to solid supports by conventional means such as covalent
or electrostatic bonding.
"Amplification reaction mixture" refers to an aqueous
solution comprising the various reagents used to amplify a
target nucleic acid. These include enzymes, aqueous buffers,
salts, target nucleic acid, and nucleoside triphosphates.
Depending upon the context, the mixture can be either a
complete or incomplete ampli ication reaction mixture.
"Amplification reaction system" refers to any in
vitro means for multiplying the copies of a target sequence of
nucleic acid. Such methods include but are not limited to PCR,
DNA ligase, QB RNA replicase and RNA transcription-based
amplification systems. These involve multiple amplification
reagents and are more fully described below.
"Amplification reaction tube(s)" refers to container
suitable for holding the amplification reagents. Generally the
tube is constructed of inert components so as to not inhibit or
interfere with the amplification system being used. Where the
system requires thermal cycling of repeated heating and
cooling, the tube must be able to withstand the cycling process
and typically precisely fit the wells of the thermocycler.
"Amplification reagents" refer to the various
buffers, enzymes, primers, nucleoside triphosphates both
conventional and unconventional, and probes used to perform the
selected amplification procedure.
"Amplifying" or "Amplification" which typically
refers to an "exponential" increase in target nucleic acid is
being used herein to describe both linear and exponential
increases in the numbers of a select target sequence of nucleic
acid.
"Bind(s) substantially" refers to complementary
hybridization between oligonucleotides and embraces minor
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WO92/11273 PCT/US91/09688
mismatches which can be accommodated by reducing the stringency
of the hybridization media to achieve the desire priming of the
PCR polymerases.
"Biotinylated" refers to a biotin moiety covalently
attached to the 5' end of an oligonucleotide for the purpose of
reacting with streptavidin in a detection assay.
~ Bound~ in the context of nucleic acids refers to the
hybridization of the nucleic acids through complementary base
pairing.
"Capture probes" refer to oligonucleotides which are -
bound to a solid support and are capable of hybridizing with
detection probes either directly or indirectly through target
nucleic acid. The capture probes may be bound to the solid
support through a variety of known means including covalent
bonds and electrostatic bonds.
"Concentration" refers to the molar amount of a
substance in a given amount of volume or space.
"Detection probes" refers to nucleic acids which
bind to capture probes. The detection probe either is directly
detectable as with a radioisotope incorporated into the
sequence or indirectly labelled as with biotin wherein a
streptavidin/enzyme complex is subsequently bound. Detection
probe is also meant to include a target nucleic acid/signal
probe complex of a ternary or quaternary sandwich hybridization
assay. Such systems typically require the target nucleic acid
to bind to the capture probe and the signal probe then binds to
the target nucleic acid.
"Discrete" refers to regions of capture nucleic acid
affixed to the solid support having boundaries which provide
separation and identification allowing one to distinguish
between other regions of capture nucleic acid.
"High stringent hybridization conditions" refers to
temperature, solute concentrations and polarity of a given
hybridization medium which provides detectable differences in
hybridization rates between oligonucleotides of between 10 and
30 bases wherein the oligonucleotides mismatch their respective
complementary bases by 1 mismatched base pair. The different
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~092/11273 ll PCT/US91/09688
hybridization rates are distinguished using the detection
system of the given assay.
"Internal positive control (IPC) oligonucleotide
sequence" refers to a recombinant or synthetic oligonucleotide
that amplifies with the same primer pair used to amplify target
nucleic acid. IPC oligonucleotide sequences are used to ensure
assay users that the amplification process has occurred in the
event that the sample being tested has no target nucleic acid.
These oligonucleotides are flanked at one or both ends by a
sequence complementary to the binding site o~ an amplification
primer used in the amplification process. The internal target
subsequence is a foreign sequence not naturally found adjacent
to the binding sites of the amplification primers. The
internal control oligonucleotide sequences thus serve as
templates for the amplification primers to establish that the
amplification reaction would have amplified target nucleic acid
if such target had been present. IPC oligonucleotide sequences
can be supplied as single or double stranded nucleotides. When
single stranded those of skill would recognize that to
facilitate non-linear amplification the 5' end of the IPC
should have base identity with the other primer of the pair.
"Legionella species" include those members of the
family Legionellaceae. It is one genus family composed of more
than 2~ species. It should be recognized that the taxonomy of -~-
prokaryotes is not static and that additional species might be
named or that these species may some day be incorporated into ~ -
an alternative family or genus. ~ -
"Nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single or double stranded form
and unless otherwise limited would encompass known analog of
natura:L nucleotides which can function in a similar manner as
naturally occurring nucleotides.
"Nucleotide polymerases" refers to enzymes able to
catalyze the synthesis of DNA or RNA from nucleoside
triphosphate precursors. In the amplification reactions of
this invention the polymerases are template dependent and
typically extend from the 3' end of the polymer being formed.
It is most preferred that the polymerase is thermostable as
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described in U.S. Patent No. 4,8a9,819 and U.S. Serial No.
7/143,141, filed January 12, 1988, now abandoned.
"Positive gradient of visual density" refers to a
response of visual density which increases with the presence of
an increased presence of detection probe. In the assays
described herein it is possible to provide a dynamic range of
concentrations wherein an increase in detection probe is
noticeable. ~3elow this range, there is little or no visible
density and above the range, increased detection probe in a
sample will not yield increased visual density.
"Primer" or "nucleic acid polymerase primer(s)~
refers to an oligonucleotide, whether natural or synthetic,
capable of acting as a point of initiation of DNA synthesis
under conditions in which synthesis of a primer extension
product complementary to a nucleic acid strand is initiated,
i.e., in the presence of four different nucleotide
triphosphates and an agent for polymerization (i.e., DNA
polymerase or reverse transcriptase) in an appropriate buffer
and at a suitable temperature. A primer is preferably an
oligodeoxyribonucleotide and is single stranded for maximum
efficiency in amplification, but may also be double stranded.
If double stranded, the primer is first treated to separate its : -
strands before being used to prepare extension products. The
exact length of a primer will depend on many factors, but
typically ranges from 15 to 25 nucleotides. Short primer
molecules generally require cooler temperatures to form
sufficiently stable hybrid complexes with the template. A
primer need not reflect the exact sequence of the template but
must be sufficiently complementary to hybridize with a
template. An example of a non-complementary sequence which may
be incorporated into the primer is a sequence which encodes a
restriction enzyme recognition site (see U.S. Patent No.
4,800,159)~ -
A primer can be labeled, if desired, by incorporating
a label detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example,
useful labels include 32p, fluorescent dyes, electron-dense
reagents, enzymes (as commonly used in FLISAS), biotin, or
.
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092/11273 PCT/US91/09688
13
haptens or proteins for which antisera or monoclonal antibodies
are available. A label can also be used to "capture" the
primer, so as to facilitate the immobilization of either the
primer or amplified DNA on a solid support.
"Proportion of the two capture probes in the blend"
refers to the relative concentrations of the probes,(i.e., 1:1
or 2:1).
"Signal probe" refers to an oligonucleotide which
comprises a detectable label. This term is generally used to
differentiated between the target nucleic acid and capture
probe in a ternary or quaternary sandwich assay format. The
label can be any of the reporter or signal systems described
for the detection probe.
"Solid support" refers to any insoluble material
which can provide a substrate upon which to affix capture
nucleic acids. Such substrates may include nylon, amino or
carboxy activated plastics, glass, cellulose and the like.
"Subsequence" refers to a sequence of nucleic acids
which comprises a part of a longer sequence of nucleic acids.
"Target region" refers to a region of a nucleic acid
which is to be analyzed, which usually contains polymorphic DNA
sequences.
"Target nucleic acid~' refers to a nucleic acid which
complexes with a signal probe to provide the detection probe.
Such nucleic acids are typically used in the sandwich assay
formats wherein the target nucleic acid binds to a capture -~
probe and the signal probe binds to the target nucleic acid.
"Test Sample" refers to an aqueous hybridization
medium containing detection probes.
"Uniform density" refers to equivalent density either
through visual recording with the eye or with an analytic
instrument such as a densitometer or colorimeter.
"Visual densityll refers to the relative intensity of
the signal or detection product produced by the labelling
system used to measure the presence or absence of duplex
formation between complementary oligonucleotides. The choice
of labelling systems is not critical. The system can be direct
or indirect and may be radioactive, fluorescent, luminescent or
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WO92~11273 14 PCT/US91/09688
colorimetric. Density may be determined either by photographic
means, analytic instruments or by the naked eye. Enzymatic
colorimetric labeling systems are preferred.
DETAILED DESCRIPTION
Introduction:
The detection of Legionella species using PCR to
amplify nucleic acid from these species requires multiple
steps. These steps include an adequate sampling procedure to
isolate and/or concentrate the extant organisms to a suitable
degree for detection, a method for lysing the cells to reIease
nucleic acid, a suitable procedure to amplify the target
nucleic acid sequences and a means to detect the amplified
sequences. A general description of this methodology can be
found in Atlas, R.M. and Bej, A.K., Detecting Bacterial
Pathogens in Environmental Water Samples by Using PCR and Gene
Probes in PCR Protocols, Ed. Innis et al., Academic Press, Inc.
l990 at pages 399-406.
To effectively amplify target nucleic acid ~; -
subsequences, PCR requires primers to initiate polymerase
extension. The selection of primers is an important aspect of
this invention and the particular primers disclosed herein
solve various problems associated with other primers purported
to function adequately for the detection of Legionella species. -
Such problems relate to the binding of primers to hairpin turns
within the genome, undue internal secondary structure within
primers themselves, non-specific binding of the primers to both
target regions and to non-target regions, unequal thermal
melting points such that the primers are not all efficiently ;
binding at the same temperature and maintaining relatively
short amplified product.
The 5S RNA gene amplification primers of this
invention are able to amplify numerous species of the
~egionella genus (see Table l). They advantageously do not
amplify closely related species from the genus Pseudomonas or
Flavobacterium. The mip primers are specific for their ability
to amplify the mip gene of L.-pneumophila, L. hackeliae and
strains of L. sainthelensi.
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~092/11273 15 PCT/US91/09688
Sampling systems:
Legionella exists in warm aquatic environments.
Human infections of pathogenic Legionella often involve aerosol
exposure to warm fresh water such as that found in air
conditioning systems and residential water supplies. For
purposes of sampling, suspect water is collected and either
subjected to low speed centrifugation (eg., 10,000 X g for 15
minutes) or filtered to concentrate or collect the ~egionella.
Legionella are trapped by filters having a porosity of about
0.45~m. A prefilteration step can be useful where physical
debris may interfere with the subsequent filtration or
amplification procedures. The prefilter is a noncritical
feature of this invention. Such filters are generally of a
porosity and composition that will allow the Legionella
bacterium to pass freely through it. The physical means for
water collection are well known and include sterile glass
vials, syringe assemblies and other inert vessels suitable for
storag~ of water.
Sample preparation:
Relatively clean water samples may be directly
assayed without isolation or purification of the target nucleic
acid prior to amplification. Large water samples may be
filtered to concentrate microbial populations. Filtering
procedures and filters are well-known to those of skill. See,
for example, Standard Method For The Examination Of Water and
Wastewater, 17th Ed., page 9-14, published by the Amer. Publ. -
Health Assoc., 1015 Fifteenth St. N.W., Washington, D.C. 20005.
Preferred filters have a porosity which will trap Legionella
cells yet permit extraneous nucleic acid to pass. Teflon
membranes such as Fluoropore FGLP 0013 of 0.2 ~M, or 0.5 ~M
pore size (Millipore Corp., Bedford, MD), or Duropore HVLP are
of use in this invention.
The water samples or filter concentrated samples are
subjected to conditions sufficient to release the target
nucleic acid from the suspect organisms. Mechanical lysis is
preferred. Mechanical lysis can be achieved by sonication,
boiling or multiple freeze/thaw cycles. Boiling the sample in
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WO92/~12~3 ~ 16 PCT/US91/09688
the presence of Chelex , resin is preferred. Chemical means of
cell disruption are also operable and include standard lysing
means such as lysozymes, osmotic shock, protease K treatment,
and detergents. Chemical methods are less preferred because of
possible detrimental effects on the PCR process, (ie.
inhibition of the Ta~ polymerase).
The samples can be heated to denature proteases and
nucleases which might interfere with the components of the PCR
reaction mixture. Heating the samples to 85C for about 5
minutes is generally sufficient. Alternatively, chemical
nuclease and protease inhibitors can be used.
When the sample is a complex mixture such as from a ~, -
patient suspected of being infected with Legionella, it may be
preferable to isolate the nucleic acid from the original
sample. A variety of techniques for extracting nucleic acids
from biological samples are known in the art. For example, see
those described in Higuchi, "Simple and Rapid Preparation of
Samples for PCR" chapter 4 in PCR Technoloqy ed. Erlich,
Stockton Press 1989); Maniatis et al., 1989, Molecular Cloning:
A Laboratory Manual, (New York, Cold Spring Harbor Laboratory,
- 1982; Hagelberg and Sykes, 1989, Nature 342:485; or Arrand, -
Preparation of Nucleic Acid Probes in Nucleic Acid
Hybridization, A Practical Approach, Ed Hames and Higgins, IRL
Press. pp. 18-30, 1985. Whole nucleic acid extraction
procedures typically invol~e an initial contacting with phenol,
phenol/chloroform or guanidinium salts, followed by an alcohol
precipitation. Genomic DNA may be obtalned from a whole
nucleic acid extraction by using Rnase before further alcohol
precipitation.
PCR procedures:
Although the PCR process is well known in the art
(see U.S. Patent Nos. 4,683,195 and 4,683,202 which are
incorporated herein by reference) and although a variety of
commercial vendors, such as Perkin-Elmer or Perkin-Elmer Cetus
instruments, sell PCR reagents and publish PCR protocols, some
general PCR information is provided below for purposes of
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~092/11273 l7 PCT/US91/09688
clarity and full understanding of the invention to those
unfamiliar with the PCR process.
To begin the PCR process, the target nucleic acid in
the sample is denatured (assuming the sample nucleic acid is
double-stranded). Denaturation is typically achieved by
heating the samples. This is because chemical denaturants may
inhibit the polymerase activity.
Once the strands are separated, the next step in PCR
involves hybridizing the separated strands with primers that
flank the target region or subsequence. The primers are then
extended to form complementary copies of the target strands,
and the cycle of denaturation, hybridization, and extension is
repeated as many times as necessary to obtain the desired
amount of amplified nucleic acid.
Template-dependent extension of primers in PCR is
catalyzed by a polymerizing agent in the presence of adequate
amounts of four deoxyribonucleotide triphosphates (dATP, dGTP,
dCTP, and dTTP) in a reaction medium comprised of the
appropriate salts, metal cations, and pH buffering system.
Suitable polymerizing agents are enzymes known to catalyze
template-dependent DNA synthesis. For example, if the template
is RNA, a suitable polymerizing agent to convert the RNA into a
complementary DNA (CDNA) sequence is reverse transcriptase
(RT), such as avian myeloblastosis virus RT. Once the target
for amplification is DNA, suitable polymerases include, for
example, E. coli DNA polymerase I or its Klenow fragment, T4
DNA polymerase, and Taq polymerase, a heat stable ~NA
polymerase isolated from Thermus aquaticus and commercially
available from PECI. The latter enzyme, Taq DNA polymerase, is
widely used in the amplification and sequencing of nucleic
acids. The reaction conditions for using DNA polymerases are
known in the art, and are described in, for example, the
treatise Methods in Enzymology, and in Maniatis et al.,
Molecular Cloning: A Laboratory Manual, supra.
During the PCR process, the temperature is very
carefully controlled so that strand separation and primer
annealing and extension occur in equilibrium. The control of
.
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WO92/11273 18 PCT/US91/09688
temperature is typically achieved using dry heat generated from
a thermocycler.
In the preferred embodiment of the PCR process, the
reaction is catalyzed by a thermostable DNA polymerase enzyme,
5 such as Taq DNA polymerase, and carried out at an elevated --
temperature. The preferred temperature is one at which the
enzyme is thermostable, and at which the nucleic acids are in
an equilibrium of single and double strands, so that sufficient
primer will anneal to template strands to allow a reasonable
rate of polymerization. Strand separation is achieved by
heating the reaction to a sufficiently high temperature for
sufficient time to cause the denaturation of the duplex, but
not to cause an irreversible denaturation of the polymerase.
The PCR method can be performed in a step-wise
fashion, where after each step new reagents are added, or in a
fashion where all of the reagents are added after a given -
number of steps. For example, if strand separation is induced
by heat, and the polymerase is heat-sensitive, then the
polymerase will have to be added after every round of strand
separation. However, if, for example, a helicase is used for
denaturation, or if a thermostable polymerase is used for
extension, then all of the reagents may be added initially, or,
alternati~ely, if molar ratios of reagents are of consequence
to the reaction, the reagents may be replenished periodically
as they are depleted by the synthetic reaction.
Those skilled in the art will know that the PCR
process is most usually carried out as an automated process
with a thermostable enzyme. In this process, the reaction
mixture is cycled through a denaturing step, a primer annealing
step, and a extension step. A DNA thermocycler, a machine
specifically adapted for use with a thermostable enzyme is
disclosed more completely in EP 236,069 and U.S. Patent No.
4,889,819. DNA Thermocyclers are commercially available from
Perkin-Elmer or Perkin Elmer Cetus Instruments (PECI), Norwalk,
Conn.
A preferred mode for carrying out the PCR reaction is
the multiplex mode. The multiplex mode involves the
simultaneous amplification of different target regions using
.
.-. . : :
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W O 92/t1273 PC~r/US91/09688 19
more than one set of PCR primer pairs. The multiplex procedure
is preferably designed around primer pairs which have similar
thermal melting points. It is preferred that all pairs have
Tms within 8 of each other and that the average Tm is between
45 and about 70C with preference for an average Tm of between
60 and 70C.
Selection of the Legionella specific primers:
The primers of this invention are provided in Table
2. The primer pairs responsible for amplifying the 5S rRNA of
Legionella species are a degenerate mixture of primers. This
mixture is valuable for ensuring that only target DNA from
Legionella 5S RNA is amplified. By comparison and empirical
analysis it was determined that the left and right primers for
the 5S RNA wlll encompass at least five species of Legionella
including the important pathogenic forms. These include L.
pneumophila, isolates Bloomington, Knoxville -1, Philadelphia-1
and Togus-1), L. pneumophila fraseri tLos Anyeles), L.
bozemanii and L. dumoffii, L. jordanis, L. gormanii, L. ~ -~
longbeachae and will not react to the DNA of common bacteria
present in water samples, ie., Flavo~acterium rigense,
Pseudomonas fluorescens and aerogenes, Proteus vulgaris, and
Escherichia coli. Table 1 provides a list of Legionella
species able to be detected using the disclosed 5S RNA primers.
These primers also have compatible Tms of about 65C and bind
to regions of target sequences having relatively low secondary
structure.
The left and right primers for the mip gene are
directed to amplify that region of the gene which is specific
for pathogenicity and able to identify the species L.
pneumophila from most other Legionella species. ~oth these
primers have Tms of about 68C and bind to highly conserved
regions of the Legionella genome that have little te~dency to ~ ~-
form secondary structures such as hairpins,
Table l. Legionella species having target 5 S RNA genes
amplified by PT82 and PT80
1. Legionella pneumophilia SG4
.
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WO 92/11273 20 PCT/US91/09688
2. L. pneumophilia SG3
3. L. pneumophila SG11
4. L. pneumophila SG6
5. L. pneumophila SG7
6. L. pneumophila SG10
7. L. longbeachae SG1
8. L. pneumophila SG2
9. L. hackeliae SG1
10. L. pneumphilia SG1
10 11. L. feeleii SG2
12. L. pneumophila SG9 ::
13. L. erythra
la L . jordanis
15. L. pneumophila SG15
15 16. L. tucsonensis
17. L. pneumophila SG12
18. L. dumoffii
19. L. pneumophila SG8
20. L. jamestowniensis
20 21. L. spiritensis
22. L. sainthelensi SG1
23. L. maceachernii
24. L. moravica
25. L. quinlivanii
2 5 26. L . cincinnatienisis
27. L. bozemanii SGl
28. L. parisiensis
29. L. feeleii SGl
30. L. parisiensis
30 31. L. feeleii SGl
3 2. L. santicrucis
33. L. pneumophila SG5
34. L. birminghamensis
35. L. cherrii
35 36. L. bozmannii SG2
37. L. micdadei
38. L. oakridgensis
39. L. gratiana
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' ~ 09~l1273 PCT/US91/09688
21
40. L. pneumophila SG14
41. L. anisa
42. L. pneumophila SG13
43. L. gormanii
5 44. L. wadsorthii
45. L. longbeachae SG2
46. L. sainthelensi SG2
10 Detection of amplified product: -
The detection of the PCR products can be accomplished
by direct visualization of the gels following ethidium bromide
staining or by indirect means using specific nucleic acid
hybridization probes. These methods are well known to those of
skill and a general review of such techniques can be found in
Nucleic Acid Hybridization, A Practical Approach, Eds. Hames
and Higgins, IRL Press, Washington, D.C. 1985.
When detection of the PCR products is by
hybridization as in a Southern blot, nucleic acid probes
specifically complementary to a subsequence of the amplified
region are used. Such probes are readily obtainable from the
sequence of the amplified segment. Probes preferably hybridize
to a DNA subsequence located between the primer binding
subsequences to avoid any overlap of primer sequences and probe
sequence. Preferred probes for the 5S rRNA and the mip gene of
Legionella are provided in Table 2 (capture probes).
Depending on the assay format, it may be helpful to
have labelled probes function to detect the amplified product.
The probes can be labeled by any of the methods known in the
art. Radioactive and enzyme labels are preferred. Most
preferred are enzyme labels which in the presence of an
appropriate substrate will produce a colored product. Examples
include horseradish peroxidase and alkaline phosphatase. Color
development can be accomplished by a variety of means using a
variety of known substrates. Preferred methods for horseradish
peroxidase include using tetramethylbenzidine (T~3) as
described in Clin. Chem. 33(8):1368-1371 (1987). An
alternative detection system is the Enhanced Chemiluminescent
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WO92/11273 PCT/US91/09688
22
(ECL) detection kit commercially available from Amersham. The
kit is used in accordance with the manufacturer~s directions.
The electrophoresing conditions and the means for
detecting the individual amplified oligonucleotides are well
5 known and are not critical aspects of this invention. Any of -
the means accepted by those of skill will be applicable for
this invention.
An alternative mode of detection of the amplified
product is by sandwich hybrldization onto membrane strips. In
the typical arrangement, the strip is made of nylon and has
oligonucleotide capture probes covalently bound in discrete
spots.
To bind oligonucleotide capture probes to nylon, they
are first tailed with oligothymidylic acid of about lO0 bases.
Tailing can be achieved using terminal transferase such as
calf-thymus terminal deoxynucleotidyl transferase or
polythymidine oligonucleotides can be chemically synthesized
alony with the probes. The tailed oligonucleotides are spotted
onto the nylon strips. The strips are dried overnight and
exposed to ultraviolet irradiation. The strips are then stored
until hybridization assays are conducted. Additional details
are provided in copending and co-assigned U.S. patent
application, Serial No. 347,495, filed May 4, l9~9 is hereby
incorporated by reference.
Capture probes are as described in tables 2 and 3 and
are able to bind to the amplified products of the Legionella
genome. Amplified product is allowed to bind to the membrane
through hybridization to the capture probes. Amplified product
is then detected by enzyme activity. A preferred means for
detection of amplified product is to biotinylate each primer so
that the amplified product is able to bind a streptavidi~-
horseradish peroxidase complex. The complex then permits
detection of amplified product by chromagen development using
commercially available substrates. Primer pairs may be
bio~inylated using the procedures described in Levenson C. and
C. Chang, Nonisotypically Labeled Probes and Primers in PCR
ProtocQls, Ed. Innis et al., Academic Press, Inc. l990 at pages
99-112. Alternatively synthesis using a phosphoramidite
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`;`~`'~092/tl273 PCT/US91/09688
23
linkage to directly biotinylate is available using reagents
from Glenn Research.
Kits:
The primers of this invention can be embodied into
kits for the detecting Legionella species in the environment.
Such kits would include a variety of components. The kits
would preferably include Legionella pneumophila DNA preferably
a known pathogenic form such as the Bloomington isolate. This
DNA will serve as a positive control. A bulk polymerase chain
reaction mix which would contain optimized concentrations of
Amplitaq DNA Polymerase, a thermal resis~ant nucleic acid
polymerase zvailable from PECI, all 5S rRNA primers, all mip
primers, dNTPs (dUTP preferably replacing dTTP), and buffer
salts. An internal positive control sequence (IPC) is included
to ensure that the PCR procedure functions properly. The IPC
comprises an amplifiable target which acts as a positive
control regardless of whether the Legionella target nucleic
acid is present. In a preferred embodiment, the IPC comprises
20 an unnatural sequence selected from a portion of the human HLA - --
DQ~ gene. The gene sequences are flanked by sequences
complementary to the upstream and downstream primers used to
amplify subsequences of either the mip or 5 S RNA genes. Human
HhA DQa is a sequence which is relatively rare in water samples -~
and not likely to be present in the sample at a detectable
level. The sequence for a preferred IPC is provided in Table
3. The IPC can be either double or single stranded and may be
stored in the amplification reaction mixture or in a separate
container. Typically, IPC is added to an amplification
reaction mixture at 1 million to 10 million copies. For
quantitation of Legionella present in the samples, one can use
lower amounts of IPC, e.g., 100 to 100,000 copies. A 25 mM
MgCl2 solution is included to start the reaction by activation
of Taq polymerase.
The kit may also comprise a means to detect the
amplified product. Although any standard detection means can
be used, such as a hybridization dot blot, agarose, or
acrylamide gel e-lectrophoresis, a preferred method uses a dip
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W092/11273 2~7~ 24 PCT/US91/09688
stick having a capture oligonucleotide which binds specifically
to the amplified target. The dip stick captures the target and
is detected by a non-isotopic detection system. For example
the primers may be biotinylated before extension and a
horseradish peroxidase/streptavidin complex used to bind the
enzyme specifically to the capture site of the amplified
target. A suitable substrate\buffer combination is 3, 3', 5,
5' tetra methylbenzidine ~TMB) in lO0 mM Sodium Citrate at pH
5Ø
Controlling visual density of nucleic acid hybridization
assays:
In developing commercial quality kits, it is
preferable to control the visual density of the detection
system. Visual density conveys positive or nega~ive results to
the user and are important features of any commercial kit. It
is the purpose of the present invention to provide a uniform
and controllable visual density for such assays.
The basic problem arises from the desire to provide
clear positive/negative signals when using stringent
hybridization conditions. Nucleic acid hybridization assays
attempting to distinguish between a defined group and less than
all the group may benefit by placin~ in one site of a solid
support, a mixture of the capture probes which individually
could not capture all the different detection probes
representing the group. Such assays will typically have a
situs on a solid support bearing the mixture and other sites
having less than all the different capture probes affixed
thereto.
Without this invention, nucleic acid hybridization
assays seeking to identify and distinguish between a group and
less than all the group would not provide uniform density of
detection when assaying samples containing similar ~ -
concentrations of detection probes representing the entire
group, a subgroup or different subgroups. The reason for the
difference in visual density arises from the difference in the
thermal melting temperatures [Tm] of the various nucleic acids
comprising the assays. This problem becomes particularly acute
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~NO92/11273 PCT/US91/09688
in groups where the distinction between members can only be
made by distinguishing between oligonucleotides having
relatively few base differences, e.g. l-5 base differences in a
30 base region. Such situations require stringent
hybridization conditions and thus differences in thermal
melting temperatures, not otherwise a problem, will lead to
sharp distinctions between visual densities of positive results
where equal concentrations of detection probes are present.
In brief this method provides for a method for
controlling visual density of detection probes bound to capture
probes in a nucleic acid hybridization assay. The assays
typically have two or more different capture probes fixed to a
solid support.
The capture probes are mixed in one site of the
support according to an empirically determined proportion. A
second site containing less than all the capture probes also
prepared wherein the concentration of capture probe is
approximately equal to the concentration of that probe present
in the mixture. This proportion is designed to provide a
uniform intensity of visual density between the two sites under
a given hybridization condition and given detection probe
concentration. The goal being to provide under stringent
conditions a uniform density of label or uniform concentration
of detection probes between the sites despite the differences
in Tm between capture probes.
To assist in the understanding of this invention, a
generic model is presented. The model is a nucleic acid
hybridization assay kit for the detection of both pathogenic
species in Genus A and for detection of the two species
individually. The target nucleic acid will be a twenty base
region of the 5S ribosomal RNA. This region will differ
between the two species in that one species has a
guanosine:cytosine instead of an adenosine:thymine pairing.
Thus the Tm for species having the extra G:C pair is higher
than for the other species.
The assay is as described herein for the Legionella
assays. A collection of capture probes are first affixed to a
solid support and PCR amplification product having
. - .
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2~7~
W092/11273 26 PCT/US91/09688
biotin/streptavidin bound enzyme labels are used as detection
probes. The array of capture probes on the solid support are
three: (l) a first situs containing a mixture of l:l of the
species specific capture probes to identify, none, either or
both members of the genus; (2) a second situs containing only
capture probes specific for species l; and (3) a third situs
containing only capture probes specific for species 2.
If the mixture of situs l is a l:l mixture, the
density of the detection probes hybridized thereto for a given
concentration of probes to in a test sample will provide
density X+Y where X~ and Y' are the relative proportion of
detection probes specific to species l and 2 in a sample. The
problem is that density X will not necessarily begin to equal Y
where concentration of X' and Y' begin to equal each other in a
sample. This is because under high stringent hybridization
conditions, the numbers of detection probes in a aqueous sample
that bind to capture probes will be different according to
their respective thermal melting points.
One solution is to dilute the capture probe having
the higher Tm to accommodate the capture probe having a lower
Tm. For example in the above generic model, it is suggested
that the mixture of capture probes to species l and 2 be
diluted l:l, 2:l, l:2, 3:l and l:3. The other sites containing
only one capture probe will be likewise diluted. A reference
solution of detection probes at a given concentration is then
used along with various dilutions to achieve a rough estimate
of the visual density of the system for a given hybridization
solution and detection/labelling system.
By further routine titration, it is possible to
-achieve a multiple dot nucleic acid hybridization system using
highly stringent hybridization conditions where the visual
density of the results from the detection system are equal for
a given concentration of detection probe regardless of the Tms
for the individual oligonucleotides. Such a system will have a
mixture of capture probes in a given proportion affixed to a
solid support and a concentration of individual capture probes
affixed to another solid support or different region of the
same solid support where the individual capture probes are
... . . - - . - . - :
.
2 ~ 7 .~ r3
'`'``:~092/11273 PCTtUS91/09688
27
present on the solid support in concentrations equivalent or
approximately equivalent to their concentration in the mixture.
It being further understood that the invention is equally
applicable to situations where the mixture of probes are more
complex than two members and the individual capture probes may
represent multiple capture probes that are less than the entire
group.
In the preferred assay format, the detection probe
will be an amplification product wherein PCR is used to amplify
a select target region of an organism. The PCR process yields
biotinylated amplification products which serve as detection ~ -
probes. These assays use a reference number of original copies
in a given sample and the sensitivity of the assays is adjusted
accordingly. For example, if a regulatory agency wants a
detection level of lO0 cells per liter and each cell has lO
copies of a target region, the assays can be designed to a
collect one liter of sample and extract the nucleic acid from
the collected cells. The lO00 targets are then amplified by 30
rounds of PCR at 95~ efficiency to yield 5xlOll copies. This
number is then converted into moles using Avogadro number.
The sample of 5xlOll copies represents the minimum
acceptable sensitivity. The sample is then used as a reference
against other cells in the group (at similar concentrations) to
define conditions yielding an acceptable visual density for
that concentration.
This process defines the dynamic range of an assay.
The dynamic range are those concentrations of detection probes
wherein the subsequent visual density is detectable and
directly responsive to increases in the concentration of the
detection probe. The dynamic range is defined empirically. It
is simply a matter of routine titration to set out acceptable
hybridization conditions and labelling systems to provide the
desired dynamic range. For the colorimetric system described
in this invention using TmB, the preferred dynamic range is
between lO x lO-l2 to about lO0 x lO-9 molar.
This invention has broad application to the field of
nucleic acid hybridization. The means for affixing nucleic
acids to solid supports, to detecting hybridized nucleic acid,
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., . : .' . . : . ., ', ' .. ' .' . ' ' .' . '' ' ' .. ' . .
2U~ -J' ~93
WO 92/l1273 PCI/US91/09688
28
to isolating, selecting and amplifying probes, and for
formatting the assay including sandwich assays of three or more
oligonucleotides and binary assays of two oligonucleotides are
well known in the art. These features are not critical to the
5 invention. For an overview of the underlying technology, see
U.S. Pat. Nos. 5,015,569 and 4,886,741. A primer on this
technology is Nucleic Acid Hybridization. A Practical A~proach,
Eds. Hames, ~3.D. and Higgins, S.J., ILR Press 19 8 7 . These
three references are incorporated herein by reference.
An example of use of blended capture probes for
development of a Legionella test kit is described in the
Example section below. A target sequence of the 5S rRNA gene
was used. The 5S RRNA nucleic acid sequence for organisms
within the genus Legionella are quite similar to those of other
15 genera (i.e., Pseudomonas and Vibrio). Within the genus
Legionella the target nucleic acid sequence is not the same for
all species. A blend of the two capture probes were made in
order to detect most of the organisms of the genus Legionella
in a single detection testing.
These two capture probes are 18 bases long and vary
by one base; one probe has a "G" base where the other has a
"A". The Tm of these two probes are approximately 60 and 58 C
respectively. A titration was done to choose the proper ration
of these capture probes and found to be in this case 1:1. A
1:1 mixture of the probes were made and the mixture was
immobilized on a membrane. This mixture capture probe is used
for the detection of the genus Legionella. This mixture makes
it possible to detect most of the species of Legionella. If
either of the probes were used alone only partial detection of
the genus Legionella would be po3sible under stringent
conditions. Under reduced stringent conditions, non-Legionella
organisms with similar 5S rRNA sequences such as organisms of
the genus Pseudomonas could also be detected and produce false
positive results.
It will be apparent to those of skill that various
substitutions and modifications may be made to the invention
disclosed herein without departing from the scope and spirit of
the invention. The following examples are provided for
.
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092/11273 PCT/US91/09688
29
illustration purposes and should not be construed as a
limitation of this invention.
EXAMPLES
A. Isolation of Sample DNA
A water sample of between lO0 to 500 ml is first
filtered through a 25 mm, .45~ pore filter (Durapore from
Millipore). The filter is transferred to a tube containing 2
ml of DNA extraction rea~ent( 20~ Chelex resin, lO mM Tris-
HCl, O.l mM EDTA pH 8.0) and vortexed to free the organismsfrom the filter surface. The sample is placed in a boiling
water bath and heated to 99C in a heat block for lO min to
lyse the organisms.
B. PCR Amplification of the Le~ionella genome
As previously stated, the preferred PCR procedure is
a multiplex reaction where both the mip and 5S rRNA target
regions are amplified simultaneously in the PCR mixture. A 20
~l sample from the above test solution is removed and added to
65 ~l of an amplification reaction mix containing the
following: PCR buffer (50 mM KCl, 50 mM Tris-HCl at pH 8.9); 4
units of Taq DNA polymerase (from PECI), 0.35 mM dNTPs-and O.S
~M of each primer. The primers are described in table 2.
Three hundred copies of the internal positive control (IPC) -
sequence is added to the reaction mixture. The IPC sequence is
provided in Table 3. The sequences extending beyond PT70 are
not amplified and can be deleted.
The target 5S rRNA gene subsequence of the Legionella .
genus and the target subsequence of the mip gene of Legionella
pneumophila are amplified using PCR procedures which follow.
~,'' .
WO92/11273 PCT/US91/09688
Table 2. Primers and Probes ~for Amplifying and Detecting
Legionella species.
Left mip primer: PT69: GCATTGGTGCCGATTTGG; Seq. ID No. 6
Right mip primer: PT70: GCTTTGCCATCAAATCTTTCTGAA; Seq. ID No. 7
Right mip primer: PT181:GTTTTGCCATCAAATCTTTTTGAA; Seq. ID No.
23
Left 5S rRNA primer: PT82: GGCGACTATAGCGRTTTGGAA; Seq. ID
No. 10
Left 5S rRNA primer: PT159: GGCGACTATAGCGGTGTGGAA; Seq. ID No.
21
Right 5S rRNA primer: PT80: GCGATGACCTACTTTCRCATGA; Seq. ID
No.9
Right 5S rRNA primer: PT157: GCGATGACCTACTTTCGCATG; Seq. ID No.
mip capture probe: PT56: TTGCTTCCGGATTAACATCT; Seq. ID No. 4
mip capture probe: PT35: CAAGGCATAGATGTTAATCCGG; Seq. ID No. 2
mip ca~ture probe: PT55: CATAGCGTCTTGCATGCCTTTAGCC; Seq. ID
No. 3
nlp capture probe: PT67: AACCGAACAGCAAATGAAAGACG; Seq. ID No.
SS rRNA capture probe: PT77: CGCGCCAATGATAGTGTGA; Seq. ID No. 8
5S rRNA capture probe: PTlC0: CATCTCGAACTCAGAAGTGAA~C; Seq. ID
No. 11
5S rRN~ capture probe: PT125: GCGCCAATGATAGTGTG; Seq. ID No. 24
30 5S rRNA capture probe: PT127: GCGCCGATGATAGTGTG; Seq. ID No. 25
**Unless stated otherwise all sequences are presented 5' to 3'.
preferred sequences
The solution is overlayed with 75 ~l of light mineral
oil (Sigma Chemical Co., St. Louis, MO), 15 ~l of 25 mM MgCl2
is added and the solution is subjected to the following thermal
profile using a 1 second setting to change tem~eratures as
rapidly as possible using the Perkin-Elmer Cetus DNA
Thermocycler:
1 second to 95C
95C for 1 Min.
30 cycles of:
1 second to 95C
95C for 1 Min.
1 second to 63C
63C for 1.5 Min. followed by
72C for 7 min and shutdown or hold at 6C.
.. : - - ~ . - : ~ . . : - .
: - . . ~ ., . :
. . . . - . - . . . - . .
092/11273 PCT/US~1/09688
31
Table 3. Internal Positive_Control Sequence (IPC) PT74 and
Capture Probes.
Seq. ID No. 1
5'-GCATTGGTGCCGATTTGGGGAAGTTTGATGGAGATGAGGAGTTCTACG
****PT69*********
.... 1.2, 1.3, 4...... ~ -
TGGACCTGGAGAGGAAGGAGACTGCCTGGCGGTGGCCTGAGTTCAGCA
... 1 probe
AATTTGGAGGTTTTGTTCAGAAAGATTTGATGGCAAAGCGTACTGCTGAATTCA-3' ~
......... .
3'*~***PT70*********~***** 5'
Positive Control - DQ~1
5'-TGAGTTCAGCAAATTTGGAG-3'; Seq. ID No. 16
.~ .
Negative Control - DQ~ 1.2, 1.3, 4 ~-
*
5'-GATG~GCAGTTCTACGTGG-3'; Seq. ID No. 17
represents a single mismatched base pair. -
C. Detection of Amplified DNA
Ten microliters of the PCR reaction mixture and 2
of a loadin~ gel are mixed and applied to each lane of a 3.0%
Nusieve0 and 1~ Seakem~ brand agarose gel (FMC Corp., Rockland,
ME) using TBE electrophoresis buffer (a9 mM, Tris ~Cl, 89 mM,
sodium borate and 1 mM EDTA at pH 8.2. The loading buffer is
250 mg/ml Ficol 400, 0.25 mg/ml Bromophenol Blue. The gel is
20 cm long and 12.5 cm wide. It is run horizontally for
approximately 2 hours at 150 volts. The contents of the gel
are then viewed under W light after ethidium bromide staining.
The sensitivity of this assay permits the detection
of 100 copies of the target regions per 10 ~l sample. Using
primers PT69, PT70 and PT181, the amplified mip region is
approximately 168 bp and the amplified 5S rRNA region is
approximately 107 bp. Under the above conditions the amount of -
amplified products are approximately equal.
Alternatively the amplified products can be captured
on a nylon membrane using capture probes as illustrated in
- ~;
- . - - . . .. , ~ . - ,.: : . ': . ': .. : , .:
W O 92/11273 PC~r/US91/09688
32
tables 2 and 3. A mixture of probes PT125 and PT127 gives
superior hybridization results when testing for unknown strains
of Legionella. The probes are bound to a Pall Biodyne-B nylon
membrane (Pall Biosupport Div., East Hills, NY).
In a preferred embodiment mixtures represented by
PT82 and PT80 are amplified. Eliminating PT 159 and 157
decreases slightly the specificity of the assay for ~.
spiritensis, sainthelensi (SG1 and SG2), and ~ulnlivanii
However the assay is now substantially decreases the undesired
amplification of rRNA from Pseudomonas sps. More specifically
the nonspecific crossreaction is reduced so that 100 copies of
a Legionella specific target nucleic acid will equal 105 to 107
cross-reacting Pseudomonas specific target.
The capture probes are optionally combined with p-[2-
hydroxy-l-naphthyl azo]-benzenesulfonic acid, Orange-ll dye
(certified), from Sigma chemical Co., St. Louis, Missouri. The
dye is diluted to 0.01% in 50 mM 3-[cyclohexylamino]-1-
propanesulfonic acid (CAPS) adjusted to pH 10.0 using sodium
hydroxide.
Nylon membrane is cut into strips having a
combination of capture probes PT125 and PT127 for 5S RNA, PT55
for the mip gene and the positive and negative control probes
depicted in table 3. Approximately 4 picomoles of each probe
are bound to the membrane in discrete spots. The membrane is
then washed with a mixture of 0.5% SDS w/v, 5X SSPE (diluted
from 20X standard saline phosphate EDTA buffer pH 7.4 as
prepared in Sambrook, Molecular Cloning A Laboratory Manual,
Cold Spring Harbor Laboratory Press, 1989) and 0.03% dextran
sulfate.
Forty ~1 of denaturation solution (1.2 N NaOH, 0.11M
EDTA) is added to the exemplified sample. A 50 ~l sample of
the denatured sample is added to approximately 3 milliliters of
hybridization solution comprising 0.5% w/v SDS in 5X SSPE. The
nylon membranes with capture probes bound thereto are each
exposed to 3 ml of hybridization solution in a water bath under
gentle rotation at 50-55C for 20 min.
The hybridization solution is decanted from the
membranes and biotinylated amplified target bound to the
. . . . . . . ... . . . .
- . ~ . , . . . , .~ .. . :
.. . . ~ . .
- : : ,, ,,, : . :
- - . - -
~VO 92/11273 PC~r/US91/09688
33
membrane is linked to horseradish peroxidase through
streptavidin. The enzyme is attached by incubating the
membranes under gentle rotation in a 50-55C water bath for 20
minutes in 3.0 ml of 2.5X SSPE with 0.10% w/v SDS and 25 ~l of
a commercially available streptavidin-horseradish peroxidase
solution (AmpliType Kit~ DQ~ DNA typing kit from Perkin Elmer
Cetus). The nylon membrane are washed once for 12 minutes at
50-55C under gentle rotation in an excess of 2.5X SSPE with
0.1~ w/v SDS as wash buffer and finally washed with shaking at
10 room temperature for 5 minutes in 2.5X SSPE with 0.1~ SDS and
again in 0.1 M sodium citrate at pH 5Ø
Color development is achieved by using 3,3', 5,5' -
tetramethylbenzidine (TmB). 0.25 ml of a TM3 stock solution of
2mg/ml in ethanol ls diluted in 5 ml of 0.5 M sodium citrate at
pH 5.0 and 1 ~l of a standard solution of hydrogen peroxide.
The membranes are incubated at room temperature for 30 minutes
in the presence of this solution in the dark. The membranes are
then rinsed twice in 10 ml of water at room te~perature and
results are recorded.
' . - . ; ' ~ ', : . . ' - . ':
.: . ,. . .. : -
. : . . :. ' , - . . ~ ., : , - :
., ~ .
r~
W092/11273 34 PCT/US91/09688
SEQUENCE LISTING
(1) GENERAL INFORMATION: :
(i) APPLICANT: Picone, Teresa
McCallum, Theresa
Zoccoli, Michael
(ii) TITLE OF INVENTION: PCR PRIMERS FOR DETECTION OF
LEGIONELLA SPECIES AND METHODS FOR
CONTROLLING VISUAL INTENSITY IN
NUCLEIC ACID HYBRIDIZATION ASSAYS
(iii) NUMBER OF SEQUENCES: 25
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Kenneth A. Weber
(B) STREET: One Market Plaza, Steuart Tower, Suite 2000
(C) CITY: San Francisco
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 94105
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOSJMS-DOS
(D) SOFTWARE: PatentIn Release #1.24 .-
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUM8ER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Weber, Kenneth A.
(B) REGISTRATION NUMBER: 31,677
(C) REFERENCE/DOCKET NUMBER: 7756-18-1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-543)9600
(B) TELEFAX: 415-543) 5043
,. - - . , . -, . -. . . : : -
- : . : - .. - .. - . .. ,, . ..
.~ .
- : . ~ :
-~ ~: . :
~ ~ 'i 5 ~ s
WO92/11273 P~T/US91/09688
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 150 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) ::OPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GCATTGGTGC CGATTTGGGG AAGTTTGATG GAGATGAGGA GTTCTACGTG GACCTGGAGA
GGAAGGAGAC TGCCTGGCGG TGGCCTGAGT TCAGCAAATT TGGAGGTTTT GTTCAGAAAG l~
ATTTGATGGC AAAGCGTACT GCTGAATTCA l-
.. ~ . . . .
... . .
. , . ., .~ , .
W092/11273 20'~93 36 PCT/US9~/99688
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CAAGGCATAG ATGTTAATCC GG
. . .................... ~ . - :
:. . , . , :
J ~ ~ ~
0 92/11273 37 PC~r/US91/09688
(2) INFORMATION FOR SEQ ID NO:3: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CATAGCGTCT TGCATGCCTT TAGCC 2
,, , . . .. . ~ . .... , -. --,, - : , . . .
- : . . . . .
- - .,
: . :
~-- -. . ,
: : ~ . .
. . ~ : . :- : : : . :
. : . .. .. ~ .
2 ~ 3 ~3
WO92/11273 38
PCT/US91/09688 ~:~
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
TTGCTTCCGG ATTAACATCT
: .-. - : , . : . . . ~ ., . -
- . - : , : . -:
. .
2 ~
0g2/11273 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
AACCGAACAG CAAATGAAAG ACG 2
.
.
. ..
': : , . . . .
.;i'3
WO92/11273 PCT~US91/09688
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ~:
GCATTGGTGC CGATTTGG l~
:.
-- . .
-
- . . - .
~r~ t~
0g2/11273 41 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer) .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:~:
GCTTTGCCAT CAAATCTTTC TGAA
~: : : - :
~ 75 ~
WO92/11273 42 PCT/VS91~09688
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CGCGCCAATG ATAGTGTGA
:
- : : . -, . :. :. .: . . .:, . :: .:, -. - : :.: .,:
. - - . : . :: : . . . : : .- ::
092/11273 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCGATGACCT ACTTTCRCAT GA 2
,:, .............. ' ~ . '' . ,,, '
.,: , ' ',, . ' ,. '' ' ~ ~'. ~ . ' ' .',
W092tll273 2a~ 44 PCT/US91/0968X
(2) INFORMATION FOR SEQ ID NO:lO:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:lO:
GGCGACTATA GCGRTTTGGA A 2
. - . . .. , . . :
. , - ,., - . . . . ., : :: : . .- . ~. . , . :: .
.. . . - ~ . . . . - - . -
: - . , ... .. - .: . : .
- . .- . ~ ,-, . ;
J ~
- ~O 92/1 1273 45 PCr/US91/09688
(2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS: ~ -
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CATCTCGAAC TCAGAAGTGA AAC 23
:-: . . . . .. .
, ~ :
2~7a~c~
~ WO92/l1273 46 PCT/US91/09688 ~
.
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS-
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CCAAATCGGC ACCAATGC
:,
t.`~`~,` 2i~ J~
;"W092/t1273 47 PCTJUS91/09688
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
TTCAGAAAGA TTTGATGGCA AAGC 2
.. ,, . ~ .. . . . . ., " .. ... ....... . .
'
:' ', ~ :
'. ' ~ ,
WQ92/11273 2 ~ 48 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TTCCAAAYCG CTATAGTCGC C
' . ', . ~ ,~ . . ' ' -' ',~ '` ' `` ''',
' ' ' ` : ' ` ' ' : ' ' .
". ' ' ` . , ` . ' , ,'' ' ~, ' " ~ ' ,
`, ' '
49 ~i 7 ~
' ~ 092/11273 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
TCATGYGAAA GTAGGTCATC GC
.
.
.. :: . . ......... . . .
:
WO92/1l273
50 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS-
~A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
TGAGTTCAGC AAATTTGGAG
- - . . , ~. : , . : -
. . -
r' ~
092/11273 51 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GATGAGCAGT TCTACGTGG l'
.
: . - . :. . - . . ., ... . ~ ,-, .
, - .. . - :.: . . . , : . - -
. ~
- . . : . . -:
- .. - . : . : :
2 ~ 7 ~
WO92/1~273 52 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
TTCCACACCG CTATAGTCGC C ` ;
- :- : . ,:: . : - - -- : . . .: : -. -
. .. , . . ... . ,. ,- . ., -, . . - -, .
2 ~
092/11273 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
CATGCGAAAG TAGGTCATCG C 2
, .,, , " . ..... ... . . ... ...
.. . - - . : .:
: . . . . . . : :
~ ~ r~
W O 92/11273 . 54 PC~r/VS91/09688
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
TTCAAAAAGA TTTGATGGCA AAAC 2
,
- .
. : . , . . : . . -
- ~ - ': ' ;' .,' ' . :' :
.
: .. . ~.............. .. .... .
,
rJ i ..
'i~JWO92/11273 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21~
GGCGACTATA GCGGTGTGGA A 2.
.. : - - : . - - , ~ ' . - : : :, :: : , .
2~ 56
WO92/]1273 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GCGATGACCT ACTTTCGCAT G 2:
..,- .... .
. . - . .. .-.. , . ... , ., : .
- : . ~ ,
~ . : -.. . . ~ . .... .
57
~WO92/11273 PCT/US91/09688
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GTTTTGCCATC AAATCTTTTT GAA 2
.. . . ... . . . . . .
- . .. -: .
~ ,. . . , . ,. - , -. . . . -
.~ ; :, . : . . ~: -. .. , -
- - : - . - : - . ... -
. . ~. .-
- - ~ : . .
- ., . ~ ::
. . . . : - . - . . .: . . . . . .
. . - : , -. -: - . : ,
. - - . ~ . . .: - - - : . . .
2~7 ~ ' 58
WO92/11273 PCTtUS91/09688
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GCGCCAATGA TAGTGTG
. ... .
... . . . ...
` ~ 092/11273 ~ 3~ PCT/US91/09688
~2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (probe)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GCGCCGATGA TAGTGTG
. -: :- , ,
-
, , ~ ' ' . ~ .
