CA2726396A1 - Compositions, methods, and kits using synthetic probes for determining the presence of a target nucleic acid - Google Patents

Compositions, methods, and kits using synthetic probes for determining the presence of a target nucleic acid Download PDF

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CA2726396A1
CA2726396A1 CA2726396A CA2726396A CA2726396A1 CA 2726396 A1 CA2726396 A1 CA 2726396A1 CA 2726396 A CA2726396 A CA 2726396A CA 2726396 A CA2726396 A CA 2726396A CA 2726396 A1 CA2726396 A1 CA 2726396A1
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hpv
nucleic acid
probes
sample
hpv16
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CA2726396C (en
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Irina Nazarenko
Dominic O'neil
Karolina PACHOWICZ
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Qiagen Gaithersburg LLC
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Qiagen Gaithersburg LLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Abstract

Compositions, methods, and kits are provided for determining the presence of a target nucleic acid in a sample using synthetic probes.

Description

COMPOSITIONS, METHODS, AND KITS USING SYNTHETIC PROBES FOR
DETERMINING THE PRESENCE OF A TARGET NUCLEIC ACID

RELATED APPLICATIONS
This application claims priority to U.S. provisional applications: 61/045,952 (filed on April 17, 2008; 61/113,841 (filed on November 12, 2008); and 61/147,862 (filed on January 28, 2009), all of which are herein incorporated in their entirety.

FIELD OF THE INVENTION
The present invention relates to compositions, methods, and kits using synthetic probes for determining the presence of a target nucleic acid in a biological sample.
BACKGROUND OF THE INVENTION

The detection and characterization of specific nucleic acid sequences and sequence changes have been utilized to detect the presence of viral or bacterial nucleic acid sequences indicative of an infection, the presence of variants or alleles of mammalian genes associated with disease and cancers, and the identification of the source of nucleic acids found in forensic samples, as well as in paternity determinations.
For example, the RNA or DNA for many microorganisms and viruses have been isolated and sequenced. Nucleic acid probes have been examined for a large number of infections. Detectable nucleic acid sequences that hybridize to complementary RNA or DNA
sequences in a test sample have been previously utilized. Detection of the probe indicates the presence of a particular nucleic acid sequence in the test sample for which the probe is specific. In addition to aiding scientific research, DNA or RNA probes can be used to detect the presence of viruses and microorganisms such as bacteria, yeast and protozoa as well as genetic mutations linked to specific disorders in patient samples. Nucleic acid hybridization probes have the advantages of high sensitivity and specificity over other detection methods and do not require a viable organism. Hybridization probes can be labeled, for example with a radioactive substance that can be easily detected.
As nucleic acid sequence data for genes from humans and pathogenic organisms accumulates, the demand for fast, cost-effective, and easy-to-use tests increases. It would be desirable to provide novel and effective methods, compositions, and kits for determining a target nucleic acid in a sample.

SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method for determining the presence of a target nucleic acid in a sample. The method comprises:
a) contacting one or more polynucleotide probes with the sample under a hybridization condition sufficient for the one or more polynucleotide probes to hybridize to the target nucleic acid in the sample to form double-stranded nucleic acid hybrids, wherein the one or more polynucleotide probes does not hybridize to a variant of the target nucleic acid; and b) detecting the double-stranded nucleic acid hybrids, wherein detecting comprises contacting the double-stranded nucleic acid hybrids with a first anti-hybrid antibody that is immunospecific to double-stranded nucleic acid hybrids, whereby detection of the double-stranded nucleic acid hybrids determines the target nucleic acid in the sample.
In another aspect of the invention, the hybridization of the nucleic acids and detection of the double-stranded nucleic acid hybrids are performed at the same time.
In a further aspect of the invention, after the double-stranded nucleic acid hybrids are contacted with a first anti-hybrid antibody that is immunospecific to double-stranded nucleic acid hybrids, a second anti-hybrid antibody is added to detect the double-stranded nucleic acid hybrids whereby detection of the double-stranded nucleic acid hybrids by these second anti-hybrid antibodies determines the presence of target nucleic acid in the sample.
In another aspect of the invention, synthetic RNA probes corresponding to more than one HPV type are used to detect for the presence of HPV infection.
In certain embodiments, the detecting further comprises providing a second anti-hybrid antibody that is immunospecific to double-stranded nucleic acid hybrids, wherein the second anti-hybrid antibody is detectably labeled.
In certain embodiments, the at least one probe and the anti-hybrid antibody are added in the same step.
The target nucleic acid is may be an HPV nucleic acid and in certain embodiments, it is a high risk HPV type and the variant is a low risk type or another high risk type HPV
nucleic acid. In certain embodiments, the hrHPV type is 16, 18 and/or 45.
In certain embodiments the one or more polynucleotide probes consist essentially of a sequence or a complement thereof selected from the group consisting of SEQ
IDNOs: 1-2026.
2 The present invention provides for a method of determing the presence of an HPV
target nucleic acid in a sample wherein if the target nucleic acid is HPV 16, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 1-162.
When the target nucleic acid is HPV 18, the the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 163-309.
When the target nucleic acid is HPV 45, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 842-974.
When the target nucleic acid is HPV 31, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 310-454.
When the target nucleic acid is HPV 33, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 455-579.
When the target nucleic acid is HPV 35, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 580-722.
When the target nucleic acid is HPV 39, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 723-841.
When the target nucleic acid is HPV 51, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 975-1120.
When the target nucleic acid is HPV 52, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 1121-1252.
When the target nucleic acid is HPV 56, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 1253-1367.
When the target nucleic acid is HPV 58, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 1368-1497.
3 When the target nucleic acid is HPV 59, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 1498-1646.
When the target nucleic acid is HPV 66, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 1647-1767.
When the target nucleic acid is HPV 68, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 1768-1875.
When the target nucleic acid is HPV 82, the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs: 1876-2026.
In certain embodiments, the one or more polynucleotide probes comprises the whole set of probes for that HPV type provided herein. In certain embodiments, the one or more polynucleotide probes consists essentially of or consists of the whole set of probes for that HPV type provided herein.
The present invention further provides probe sets of SEQ ID NO: 1-162 (HPV
16);
163-309(HPV 18); 842-974(HPV 45); 310-454(HPV 31); 455-579(HPV 33); 580-722(HPV
35); 723-841(HPV 39); 975-1120(HPV 51); 1121-1252(HPV 52); 1253-1367(HPV 56);
1368-1497(HPV 58); 1498-1646(HPV 59); 1647-1767(HPV 66); 1768-1875(HPV 68);
and 1876-2026(HPV 82).
The present invention further provides probe sets of SEQ ID NO: 1-161 (HPV
16);
163-299 (HPV 18); and 842-968 (HPV 45). In certain embodiments the one or more polynucleotide probes is a mixture of probe sets comprising the probes set forth in SEQ ID
NO: 1-2026.
In certain embodiments the one or more polynucleotide probes is a mixture of probe sets comprising the probes set forth in SEQ ID NO: 1-19, 21-23, 25-53, 55-65, 67-71, 73-92, 94-116, 118-130, 132-241, 244-274, 276, 277, 279, 280, 282-849, 851-893, 895-917, 919-929, 931, 933-936, 938-2026.
In certain embodiments the hybridization is performed at about 45 to about 55 C.
The present invention also provides kits comprising any one of the probes disclosed herein from SEQ ID NO: 1-2026. In certain embodiments the kits comprise the probes set forth from the group consisting of SEQ ID NO: 1-162 (HPV 16); 163-309(HPV 18);

974(HPV 45); 310-454(HPV 31); 455-579(HPV 33); 580-722(HPV 35); 723-841(HPV
39);
4
5 PCT/US2009/041033 975-1120(HPV 51); 1121-1252(HPV 52); 1253-1367(HPV 56); 1368-1497(HPV 58);

1646(HPV 59); 1647-1767(HPV 66); 1768-1875(HPV 68); and 1876-2026(HPV 82). In another embodiment, the kit comprises the probes set forth in SEQ ID NO: 1-161 (HPV 16);
163-299 (HPV 18); and 842-968 (HPV 45). In another embodiment, the kit comprises the probes set forth in SEQ ID NO: 1-2026. In yet another embodiment, the kit comprises the 2,007 probes set forth in SEQ ID NO: 1-19, 21-23, 25-53, 55-65, 67-71, 73-92, 94-116, 118-130, 132-241, 244-274, 276, 277, 279, 280, 282-849, 851-893, 895-917, 919-929, 931, 933-936, 938-2026. Advantages and benefits of the present invention will be apparent to one skilled in the art from reading this specification.

BRIEF DESCRIPTION OF THE FIGURES
Figure la shows the sequence conservation across 20 HPV genomes.
Figure lb shows location of RNA probes along HPV 18 genome.
Figure 2 shows performance of RNA probes specific for HPVs 16, 18, 31, or 45.
Figure 3 shows detection of 5,000 copies of HPV 18 plasmid with synRNA
coverage of 3.7Kb. synRNA = ((1.5kb coverage; 30mers) or (3.7kb coverage; 25mers)) @
1.34 nM
Figure 4 shows that increasing the concentration of synRNA increased sensitivity of detection.
Figure 5 shows that 50mer synRNA gave higher signal than 25mer synRNA; synRNA
= 0.5kb of coverage; 25 or 50mers @ concentrations listed above; at about 40 min hybridization @ about 50 C.
Figure 6 shows the effect of contiguous synRNA coverage on sensitivity of detection;
40 min hybridization @ 50 C; synRNA = 1.5kb of coverage; 30 mers @ 2.24 nM.
Figure 7 shows HPV16 and HPV18 detection with synRNA is comparable; 55 C
hybridization; synRNA = 3.7kb (coverage for HPV 18) or 3.175kb (coverage for HPV 16);
25 mers @ 1.34 nM.
Figure 8 shows comparison of synRNA prepared by different chemistries.
Figure 9 shows hybridization of synRNAs at different temperatures; synRNA =
3.7kb of coverage; 25mers @ 1.34 nM.
Figure 10 shows detection in the presence or absence of exogenous RNase A.
Figure 11 shows sensitivity of detection.
Figure 12 shows amplification time course.
Figure 13 shows enhancing sensitivity by increasing target amplification.
Figure 14 shows specificity.

Figure 15 represents another embodiment of a method in accordance with the present invention.
Figure 16 shows that diluting the sample collected in PreservCyt with a suitable collection medium ("DCM" - Digene Collection Medium) enhances the signal.
Figure 17 shows that synRNA probes have the same signal and dynamic range as the full length probes.
Figure 18 shows that synRNA probes detected all specific targets (15 hrHPV
target nucleic acids) with robust S/N and low variability.
Figure 19 shows that even with 108 copies of low-risk HPV mixed with 108 copies of positive control, the mixture of 2,007 hrHPV probes were specific enough not to provide a positive signal for the low risk HPV types and were still able to provide a strong signal for the positive control.
Figures 20A and B shows that decreasing hybridization temperature increases the detection signal where the biological sample containing the target nucleic acid has been collected in PreverveCyt .

DETAILED DESCRIPTION
The present inventors have discovered novel methods, compositions, and kits using synthetic probes for determining the presence of a target nucleic acid in a biological sample.
The present invention also provides synthetic probes useful for detecting a target nucleic acid in a sample. The present invention includes use of novel detection methods, compositions, and kits for, among other uses, clinical diagnostic purposes, including but not limited to the detection and identification of pathogenic organisms.
In one aspect, the present invention provides a method for determining the presence of a target nucleic acid in a sample, the method comprising:
a) contacting one or more polynucleotide probes with the sample under a hybridization condition sufficient for the one or more polynucleotide probes to hybridize to the target nucleic acid in the sample to form double-stranded nucleic acid hybrids, wherein the one or more polynucleotide probes does not hybridize to a variant of the target nucleic acid; and b) detecting the double-stranded nucleic acid hybrids, wherein detecting comprises contacting the double-stranded nucleic acid hybrids with a first anti-hybrid antibody that is immunospecific to double-stranded nucleic acid hybrids, whereby detection of the double-stranded nucleic acid hybrids determines the target nucleic acid in the sample.
6 The sample includes, without limitation, a specimen or culture (e.g., microbiological and viral cultures) including biological and environmental samples. Biological samples may be from an animal, including a human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. Particularly preferred are biological samples including, but not limited to cervical samples (e.g., a sample obtained from a cervical swab), blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen. The sample may comprise a single-or double-stranded nucleic acid molecule, which includes the target nucleic acid and may be prepared for hybridization analysis by a variety of methods known in the art, e.g., using proteinase K/SDS, chaotropic salts, or the like. These examples are not to be construed as limiting the sample types applicable to the present invention.
For example, a sample such as blood or an exfoliated cervical cell specimen can be collected and subjected to alkaline pH to denature the target nucleic acid and, if necessary, nick the nucleic acid that may be present in the sample. The treated, or hydrolyzed, nucleic acids can then be subjected to hybridization with a probe or group of probes diluted in a neutralizing buffer.
In certain embodiments, the sample is an exfoliated cell sample, such as an exfoliated cervical cell sample. The sample can be collected with a chemically inert collection device such as, but not limited to, a dacron tipped swab, cotton swap, cervical brush, etc. The sample and collection device can be stored in a transport medium that preserves nucleic acids and inhibits nucleases, for example in a transport medium comprising a chaotropic salt solution, a detergent solution such as sodium dodecyl sulfate (SDS), preferably 0.5% SDS, or a chelating agent solution such as ethylenediaminetetraacetic acid (EDTA), preferably 100 mM, to prevent degradation of nucleic acids prior to analysis. In certain embodiments, the sample is a cervical cell sample and in this situation, both the cell sample and the collection device are stored in the chaotropic salt solution provided as the Sample Transport Medium TM
in the digene Hybrid Capture 2 High-Risk HPV DNA Test kit (Qiagen Gaithersburg, Inc., Gaithersburg, MD ). Alternatively, the sample can be collected and stored in a base hydrolysis solution, for example.
The sample may be collected and stored in a liquid based cytology collection medium such as, but not limited to, PreservCyt and SurepathTM. When such collection mediums are
7 used (methanol based), it is preferable that the sample is diluted prior to performing methods of the present invention relating to detecting at target nucleic acid to obtain a stronger detection signal. A suitable solution is one that dilutes the methanol concentration, but still allows the rest of the reaction to proceed (i.e. allows hybridization of the probe to the target nucleic acid, allows binding of the hybrid capture antibody to the DNA:RNA, etc.). A useful solution is a collection medium comprising NP-40, sodium deoxycholate, Tris-HC1, EDTA, NaCl and sodium azide. In certain embodiments, the medium comprises or consists essentially of 1% NP-40, 0.25% sodium deoxycholate, 50mM Tris-HC1, 25 mM EDTA, mM NaCl and 0.09% sodium azide. This medium is often referred to herein and in the figures as Digene Collection Medium or DCM. Figure 16 shows that diluting a methanol based collection medium, such as PreserveCyt (or noted as "PC" in the figure) with a suitable solution such as DCM, produces a stronger signal and as such signals and hence detection of a target nucleic acid can be obtained even when the target nucleic acid has been collected in a relatively large volume of solution (i.e. > lml). Preferably the methanol based collection medium or PreserveCyt is diluted in the following ratios of PC to DCM:
Amount of PreserveCyt Amount of Digene (PC) in ml Collection Medium (DCM) in 1 1 about 100 to about 1500 1 about 200 to about 1300 1 about 300 to about 1200 1 about 400 to about 1100 1 about 500 to about 1000 1 about 600 to about 1000 1 about 600 to about 900 1 about 600 to about 800 In other embodiments 1 ml of PC is diluted with at least 200 l of DCM, in other embodiments, 1 ml of PC is diluted with at least 300 l of DCM, and in other embodiments, 1 ml of PC is diluted with at least 500 l of DCM. In certain embodiments, 1 ml of PC is diluted with at least 500 DCM but no more than 1000 l DCM. By diluting the PC
containing the biological sample, the methods of the present invention are able to provide results and detect a target nucleic acid from a relative large sample volume (i.e. a biological sample collected in > 1 ml).
If the nucleic acids to be determined are present in blood, a blood sample can be collected with a syringe, for example, and the serum separated by conventional methods.
8 Preferably, serum is incubated for approximately 20 minutes at approximately 65 C with a protease, such as proteinase K prior to a base treatment.
In some embodiments, the sample is treated with a base, or hydrolyzed, to render the target nucleic acid accessible to hybridization. Nucleic acids can be denatured and, if necessary, nicked by incubating the sample and collection device, if present, in 0.1 to 2.0 M
base at about 20 to about 100 C for 5 to 120 minutes. Preferably, treatment is achieved with 0.2 to 0.8 M NaOH, or a similar base such as KOH, at 60-70 C for 30 to 60 minutes. Most preferably, the sample and swab are incubated in 0.415 M NaOH for 65 C for 45 minutes.
Approximately one volume of sample can be treated with about one-half volume of base, also referred to herein as the hydrolysis reagent. The pH will typically be about 13. This basic pH will both nick and denature a majority of the nucleic acid in the specimen.
In addition, base treatment can disrupt interactions between peptides and nucleic acids to improve accessibility of the target nucleic acid and degrade protein. Base treatment effectively homogenizes the specimen to ensure reproducibility of analysis results for a given sample.
Base treatment also can reduce the viscosity of the sample to increase kinetics, homogenize the sample, and reduce background by destroying any existing DNA-RNA or RNA-RNA
hybrids in the sample. Base treatment also can help inactivate enzymes such as RNAases that may be present in the sample.
The variant of the target nucleic acid includes genetic variants of the target. A variant includes polymorphisms, mutants, derivatives, modified, altered, or the like forms of the target nucleic acid. By way of example with respect to a human papillomavirus (HPV), variants include the various types. Thus, for example, wherein the target nucleic acid corresponds to HPV type 18 nucleic acid, the variant can be a corresponding nucleic acid sequence of a type of HPV other than type 18.
In one embodiment, the target nucleic acid is an HPV nucleic acid. In another embodiment, the HPV nucleic acid is HPV DNA of an HPV type. In some embodiments, the HPV type is HPV 18, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 16, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
9 In other embodiments, the HPV type is HPV 45, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 31, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 33, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 35, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 39, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 51, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 52, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 56, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 58, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 59, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 66, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33,34,35,39,40,42,43,44,45,51,52,53,54,56,58,59,61,62,67,68,69,70,71,72,73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 68, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
In other embodiments, the HPV type is HPV 82, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 83, 84, and 89.
In other embodiments, the HPV type is HPV 16, 18 and 45, wherein the variant is nucleic acid of a low risk HPV type.
In other embodiments, the HPV type is a high risk HPV type (hrHPV), wherein the variant is nucleic acid of a low risk HPV type.
In other embodiments, the HPV type is 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 wherein the wherein the variant is nucleic acid of a low risk HPV type (such as 1, 2, 3, 4, 5, 6, 8, 11, 13, 26, 30, 34, 53, 54, 61, 62, 67, 69, 70, 71, 72, 73, 74, 81, 83, 84, and 89).
Thus, the present invention provides methods, compositions, and kit for determining a target nucleic acid in a sample. The sample can be collected with a chemically inert device and optionally treated with a base or other denaturing solution. The sample is incubated with one or more polynucleotide probes that are specific for the target nucleic acid but not for any other member of the population (i.e. will not bind to a variant). For example, the target nucleic acid to be determined can be an oncogenic or non-oncogenic HPV DNA
sequence, HBV DNA sequence, Gonorrhea DNA, Chlamydia DNA, or other pathogen DNA or RNA.
The target nucleic acid may be from cells for the detection of cancer.

In one embodiment, the target nucleic acid is an HPV nucleic acid, wherein the target and the variant nucleic acids correspond to an HPV high risk or low risk type.
HPV types characterized as low risk and high risk are known to one of ordinary skill in the art. Presently HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 82 are considered hrHPVs and HPV types 1, 2, 3, 4, 5, 6, 8, 11, 13, 26, 30, 34, 53, 54, 61, 62, 67, 69, 70, 71, 72, 73, 74, 81, 83, 84, and 89 are considered low risk HPVs.
Thus, for example, the target nucleic acid to be determined can be nucleic acid of a microorganism such as, e.g., a disease-causing pathogen, preferably a virus or bacteria, preferably HPV, however, the invention is not restricted thereto and the description following is merely illustrated by reference to determining an HPV DNA in a sample.

Polynucleotide probes ("synprobes") In accordance with the present invention, one or more polynucleotide probes are contacted with the sample under conditions sufficient for the one or more polynucleotide probes to hybridize to the target nucleic acid in the sample to form double-stranded nucleic acid hybrids. In certain embodiments, the target nucleic acid is DNA and the probes are RNA. In certain embodiments the RNA probes are short probes as opposed to full length transcribed RNA probes. These short probes are often referred to herein as synthetic RNA
probes or "synRNA."
In certain embodiments, sets of polynucleotide probes are used (i.e. more than one probe). For example, if the target nucleic acid to be detected is HPV 16, a set of probes designed to specifically (i.e. only) bind to HPV 16 as opposed to binding to other HPV types is used. In certain embodiments a set of probes is used to ensure coverage of about 3-4 kb of the target nucleic acid, which ensures a strong, readable signal. In certain embodiments, detection of HPV 16 using the methods of the present invention may use a probe set comprising all of the HPV 16 probes disclosed herein (see Table 1). In other embodiments, a set of probes designed to specifically bind to another HPV type is used. For example, for HPV 18, the set of probes comprises the probes disclosed in Table 2, for HPV
45- the set of probes comprises the probes disclosed in Table 3; for HPV 31 - the set of probes comprises the probes disclosed in Table 4; for HPV 33 - the set of probes comprises the probes disclosed in Table 5; for HPV 35 - the set of probes comprises the probes disclosed in Table 6; for HPV 39 - the set of probes comprises the probes disclosed in Table 7;
for HPV 51 -the set of probes comprises the probes disclosed in Table 8 ; for HPV 52 - the set of probes comprises the probes disclosed in Table 9; for HPV 56 - the set of probes comprises the probes disclosed in Table 10; for HPV 58 - the set of probes comprises the probes disclosed in Table 11; for HPV 59 - the set of probes comprises the probes disclosed in Table 12; for HPV 66 - the set of probes comprises the probes disclosed in Table 13; for HPV
68 - the set of probes comprises the probes disclosed in Table 14; for HPV 15 - the set of probes comprises the probes disclosed in Table 15.
In certain embodiments a probe mixture comprising multiple sets of probes is used to simultaneously screen for any one of a mixture of desired target nucleic acids. For example, it may be desirable to screen a biological sample for the presence of any hrHPV type. In such a situation, a probe mixture of some, and in some cases, all of the probes provided in Tables 1-15 are used. For example, a probe mixture can be designed to provide a probe set for every high risk HPV (hrHPV) so one test can be run to identify whether the sample had any hrHPV
target nucleic acid. For example, a probe mixture of 2,007 type-specific probes for the detection of 15 hrHPV types was used and was able to detect 5,000 copies/assay of each target genome (see Figures 17 and 18). Figure 17 shows that the synthetic probes have the same signal and dynamic range as traditional full length probes. Figure 19 provides the results of an analytical specificity test, which shows a good signal for the positive control having 108 copies, whereas the low risk HPV types had a signal below the cutoff, even when they were present at 108 copies. Thus, figures 17-19 show that the methods of the present utilizing the synthetic RNA probes ("synRNA") of the invention provide analytical specificity and are equivalent in limit of detection and dynamic range to full-length transcribed probes and do not suffer any loss of sensitivity with clinical samples. The probes of the present invention enable sensitive detection of a set of target genomes, while also achieving excellent specificity against even very similar related species. For example, the methods of the invention using the synprobes are able to distinguish HPV 67 from HPV 52 and 58 (HPV67 is greater than 72% identical to HPV 52 and 56). See Figure 19.
If a positive signal is obtained in the example above, it may then be desirable to further test the sample to identify the actual hrHPV type target nucleic acid present. In such a situation, the sample would be further tested with one probe specific for the HPV type or a set of probes for the specific HPV type. For example, if one were testing the sample to determine whether the sample contained an HPV 16 target nucleic acid, then at least one probe from Table 1 (HPV 16 probes) would be used, or alternatively the entire set of probes from Table 1 would be used to increase the signal strength. Alternatively, it may be desirable to test for certain hrHPV types such as HPV 16, 18 and 45 and not necessarily test for each individual hrHPV types. In this situation, the mixture of probes would employ at least one probe from the HPV 16, 18 and 45 probe sets (or alternatively, all of the probes from the 16, 18 and 45 HPV probe sets are used).
The one or more polynucleotide probes are designed so that they do not hybridize to a variant of the target nucleic acid under the hybridization conditions utilized. The number of different polynucleotide probes employed per set can depend on the desired sensitivity.
Higher coverage of the nucleic acid target with the corresponding polynucleotide probes can provide a stronger signal (as there will be more DNA-RNA hybrids for the antibodies to bind).
In one embodiment, the method further comprises determining the one or more polynucleotide probes, wherein determining comprises identifying a contiguous nucleotide sequence of the target nucleic acid, wherein the contiguous nucleotide sequence is not present in the variant. By way of example, relatively short regions (e.g., about 25mers) of the HPV
genome with sufficient sequence specificity can be determined to provide the one or more polynucleotide probes for HPV type-specific hybridization.
Thus, depending on the target nucleic acid of interest, and the corresponding variant(s), the one or more polynucleotide probes can be prepared to have lengths sufficient to provide target-specific hybridization. In some embodiments, the one or more polynucleotide probes each have a length of at least about 15 nucleotides, illustratively, about 15 to about 1000, about 20 to about 800, about 30 to about 400, about 40 to about 200, about 50 to about 100, about 20 to about 60, about 20 to about 40, about 20 to about 20 and about 25 to about 30 nucleotides. In one embodiment, the one or more polynucleotide probes each have a length of about 25 to about 50 nucleotides. In certain embodiments, the probes have a length of 25 nucleotides. In certain embodiments, all of the probes in a set will have the same length, such as 25 nucleotides, and will have very similar melting temperatures to allow hybridization of all of the probes in the set under the same hybridization conditions.
Bioinformatics tools can be employed to determine the one or more polynucleotide probes. For example, Oligoarray 2.0, a software program that designs specific oligonucleotides can be utilized. Oligoarray 2.0 is described by Rouillard et al., Nucleic Acids Research, 3 1: 3057-3062 (2003), which is incorporated herein by reference.
Oligoarray 2.0 is a program which combines the functionality of BLAST (Basic Local Alignment Search Tool) and Mfold (Genetics Computer Group, Madison, WI).
BLAST, which implements the statistical matching theory by Karlin and Altschul (Proc.
Natl. Acad.
Sci. USA 87:2264 (1990); Proc. Natl. Acad. Sci. USA 90:5873 (1993), is a widely used program for rapidly detecting nucleotide sequences that match a given query sequence One of ordinary skill in the art can provide a database of sequences, which are to be checked against, for example HPV high risk and low risk types 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89. The target sequence of interest, e.g. HPV
18, can then be BLASTed against that database to search for any regions of identity. Melting temperature (Tm) and %GC can then be computed for one or more polynucleotide probes of a specified length and compared to the parameters, after which secondary structure also can be examined. Once all parameters of interest are satisfied, cross hybridization can be checked with the Mfold package, using the similarity determined by BLAST. The various programs can be adapted to determine the one or more polynucleotide probes meeting the desired specificity requirements. For example, the parameters of the program can be set to prepare polynucleotides of 25nt length, Tin range of 55-95 C, a GC range of 35-65%, and no secondary structure or cross-hybridization at 55 C or below.
Accordingly in other aspects, the present invention utilizes bioinformatics to provide sequence information sufficient to design and/or prepare polynucleotide probes for determining the target in the sample.
In addition to using the synprobes in a method of the present invention, one aspect of the invention comprises the probes disclosed herein.
In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 16 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs: 1-162 (See Table 1).
In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 16, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 1-162. In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 16, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of. SEQ ID NOs: 1-161. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to comprising SEQ ID NOs: 1-162. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to comprising SEQ ID NO: 1-19, 21-23, 25-53, 55-65, 67-71, 73-92, 94-116, 118-130, 132-162.
Table 1: Polyribonucleotide probes for determining HPV 16 nucleic acid.

SEQ ID
NO: Name Sequence 1 HPV16 25 HR&LR 7866 GGGUUACACAUUUACAAGCAACUUA
2 HPV16 25 HR&LR 7841 ACAUGGGUGUGUGCAAACCGAUUUU
3 HPV16 25 HR&LR 7799 CUGUGUAAAGGUUAGUCAUACAUUG
4 HPV16 25 HR&LR 7774 AAUGUCACCCUAGUUCAUACAUGAA
HPV16 25 HR&LR 7749 AGGUUUAAACUUCUAAGGCCAACUA
6 HPV16 25 HR&LR 7712 GGCUUGUUUUAACUAACCUAAUUGC
7 HPV16 25 HR&LR 7676 CAACGCCUUACAUACCGCUGUUAGG
8 HPV16 25 HR&LR 7629 CUGAAUCACUAUGUACAUUGUGUCA
9 HPV16 25 HR&LR 7577 GCACUGCUUGCCAACCAUUCCAUUG
HPV16 25 HR&LR 7552 UGCCAAAUCCCUGUUUUCCUGACCU
11 HPV16 25 HR&LR 7527 UUGUACGUUUCCUGCUUGCCAUGCG
12 HPV16 25 HR&LR 7502 CUAUGUCAGCAACUAUGGUUUAAAC
13 HPV16 25 HR&LR 7433 CCCAUUUUGUAGCUUCAACCGAAUU
14 HPV16 25 HR&LR 7408 AUAUACUAUAUUUUGUAGCGCCAGG
HPV16 25 HR&LR 7371 UAUAAACUAUAUUUGCUACAUCCUG
16 HPV16 25 HR&LR 7340 CCUACUAAUUGUGUUGUGGUUAUUC
17 HPV16 25 HR&LR 7293 GUGUAACUAUUGUGUCAUGCAACAU
18 HPV16 25 HR&LR 7250 UGUAUGGUAUAAUAAACACGUGUGU
19 HPV16 25 HR&LR 7225 AUAUUAAGUUGUAUGUGUGUUUGUA
HPV16 25 HR&LR 7201 GUAUGUGCUUGUAUGUGCUUGUAAA
21 HPV16 25 HR&LR 7175 UAGUGUUGUUUGUUGUGUAUAUGUU
22 HPV16 25 HR&LR 7150 UGUAAGUAUUGUAUGUAUGUUGAAU
23 HPV16 25 HR&LR 7112 AUCUACCUCUACAACUGCUAAACGC
24 HPV16 25 HR&LR 7087 AACGAAAAGCUACACCCACCACCUC
HPV16 25 HR&LR 7061 GGCCAAACCAAAAUUUACAUUAGGA
26 HPV16 25 HR&LR 6935 AGCACCUAAAGAAGAUGAUCCCCUU
27 HPV16 25 HR&LR 6894 UUUGUAACCCAGGCAAUUGCUUGUC
28 HPV16 25 HR&LR 6869 AGGCACACUAGAAGAUACUUAUAGG
29 HPV16 25 HR&LR 6790 CAGACGUUAUGACAUACAUACAUUC
HPV16 25 HR&LR 6675 GCCAUAUCUACUUCAGAAACUACAU
31 HPV16 25 HR&LR 6541 CUGAUGCCCAAAUAUUCAAUAAACC
32 HPV16 25 HR&LR 6496 CCAGUUCAAAUUAUUUUCCUACACC
33 HPV16 25 HR&LR 6471 GGCUCUGGGUCUACUGCAAAUUUAG
34 HPV16 25 HR&LR 6438 GGUGAAAAUGUACCAGACGAUUUAU
HPV16 25 HR&LR 6350 GUCAGAACCAUAUGGCGACAGCUUA
36 HPV16 25 HR&LR 6294 GUUCCACUGGAUAUUUGUACAUCUA
37 HPV16 25 HR&LR 6192 CCACCAUUAGAGUUAAUAAACACAG
38 HPV16 25 HR&LR 6165 AAUGUUGCAGUAAAUCCAGGUGAUU
39 HPV16 25 HR&LR 6052 CAGGUGUGGAUAAUAGAGAAUGUAU
HPV16 25 HR&LR 6022 CAGAAAAUGCUAGUGCUUAUGCAGC
41 HPV16 25 HR&LR 5851 UAUUUAGAAUACAUUUACCUGACCC
42 HPV16 25 HR&LR 5825 UAAAGUAUCAGGAUUACAAUACAGG
43 HPV16 25 HR&LR 5800 CUAACAAUAACAAAAUAUUAGUUCC
44 HPV16 25 HR&LR 5745 GCAGGAACAUCCAGACUACUUGCAG
HPV16 25 HR&LR 5586 GUUAUUACAUGUUACGAAAACGACG
46 HPV16 25 HR&LR 5546 ACAAUUAUUGCUGAUGCAGGUGACU
47 HPV16 25 HR&LR 5521 UAUAGUUCCAGGGUCUCCACAAUAU
48 HPV16 25 HR&LR 5496 CUGACCAAGCUCCUUCAUUAAUUCC
49 HPV16 25 HR&LR 5469 CAGGUCCUGAUAUACCCAUUAAUAU

50 HPV16 25 HR&LR 5442 GUGGUGCAUACAAUAUUCCUUUAGU
51 HPV16 25 HR&LR 5406 CAGGUUAUAUUCCUGCAAAUACAAC
52 HPV16 25 HR&LR 5381 CCAUCUGUACCCUCUACAUCUUUAU
53 HPV16 25 HR&LR 5356 UACAGAUACUUCUACAACCCCGGUA
54 HPV16 25 HR&LR 5336 AUUUAUGCAGAUGACUUUAUUACAG
55 HPV16 25 HR&LR 5301 CCUCACCUACUUCUAUUAAUAAUGG
56 HPV16 25 HR&LR 5276 ACAUAUACUACCACUUCACAUGCAG
57 HPV16 25 HR&LR 5228 ACUAUUGAUCCUGCAGAAGAAAUAG
58 HPV16 25 HR&LR 5182 UGGAAAAUCUAUAGGUGCUAAGGUA
59 HPV16 25 HR&LR 5153 GGUAAUAAACAAACACUACGUACUC
60 HPV16 25 HR&LR 5122 UAGGCGUACUGGCAUUAGGUACAGU
61 HPV16 25 HR&LR 5051 AAUAGUAUUAAUAUAGCUCCAGAUC
62 HPV16 25 HR&LR 5000 GCAUAUGAAGGUAUAGAUGUGGAUA
63 HPV16 25 HR&LR 4965 CCACUCCCACUAAACUUAUUACAUA
64 HPV16 25 HR&LR 4910 GGAUUAUAUAGUCGCACAACACAAC
65 HPV16 25 HR&LR 4854 CUAACACAGUAACUAGUAGCACACC
66 HPV16 25 HR&LR 4829 GAUACAUUUAUUGUUAGCACAAACC
67 HPV16 25 HR&LR 4771 GCAUUUUACACUUUCAUCAUCCACU
68 HPV16 25 HR&LR 4706 CAUAAUAAUCCCACUUUCACUGACC
69 HPV16 25 HR&LR 4681 UAAUACUGUUACUACUGUUACUACA
70 HPV16 25 HR&LR 4640 ACUACUUCAACUGAUACCACACCUG
71 HPV16 25 HR&LR 4588 UGCACCAACAUCUGUACCUUCCAUU
72 HPV16 25 HR&LR 4562 GAAGAAACUAGUUUUAUUGAUGCUG
73 HPV16 25 HR&LR 4480 UACAGAUACACUUGCUCCUGUAAGA
74 HPV16 25 HR&LR 4435 CGGACGCACUGGGUAUAUUCCAUUG
75 HPV16 25 HR&LR 4369 AUUACAAUAUGGAAGUAUGGGUGUA
76 HPV16 25 HR&LR 4275 CGGCUACCCAACUUUAUAAAACAUG
77 HPV16 25 HR&LR 4232 ACAAUGCGACACAAACGUUCUGCAA
78 HPV16 25 HR&LR 4131 AAUUGUUGUAUACCAUAACUUACUA
79 HPV16 25 HR&LR 4103 AUAUGUACAUAAUGUAAUUGUUACA
80 HPV16 25 HR&LR 4009 CUCUGCGUUUAGGUGUUUUAUUGUA
81 HPV16 25 HR&LR 3984 UAUUACUAUUGUGGAUAACAGCAGC
82 HPV16 25 HR&LR 3942 UGCUUUUGUCUGUGUCUACAUACAC
83 HPV16 25 HR&LR 3866 UGCAUCCACAACAUUACUGGCGUGC
84 HPV16 25 HR&LR 3824 CAGUGUCUACUGGAUUUAUGUCUAU
85 HPV16 25 HR&LR 3765 UGAUAGUGAAUGGCAACGUGACCAA
86 HPV16 25 HR&LR 3712 CAUUGGACAGGACAUAAUGUAAAAC
87 HPV16 25 HR&LR 3686 UGUAUACUGCAGUGUCGUCUACAUG
88 HPV16 25 HR&LR 3638 CUAAUACUUUAAAAUGUUUAAGAUA
89 HPV16 25 HR&LR 3602 GUAACACUACACCCAUAGUACAUUU
90 HPV16 25 HR&LR 3577 CACAAAGGACGGAUUAACUGUAAUA
91 HPV16 25 HR&LR 3552 AAUCCUCACUGCAUUUAACAGCUCA
92 HPV16 25 HR&LR 3520 UUGUUGCACAGAGACUCAGUGGACA
93 HPV16 25 HR&LR 3495 CGGAAACCCCUGCCACACCACUAAG
94 HPV16 25 HR&LR 3460 ACGACUAUCCAGCGACCAAGAUCAG
95 HPV16 25 HR&LR 3417 GACCCAUACCAAAGCCGUCGCCUUG
96 HPV16 25 HR&LR 3378 UGAAAUUAUUAGGCAGCACUUGGCC
97 HPV16 25 HR&LR 3323 GUCAGGUAAUAUUAUGUCCUACAUC
98 HPV16 25 HR&LR 3241 GGAAUACGAACAUAUUUUGUGCAGU
99 HPV16 25 HR&LR 3201 GGGUCAAGUUGACUAUUAUGGUUUA
100 HPV16 25 HR&LR 3176 AAGAAGCAUCAGUAACUGUGGUAGA

101 HPV16 25 HR&LR 3145 UAUACAAACUGGACACAUAUAUAUA
102 HPV16 25 HR&LR 3103 GUGGAAGUGCAGUUUGAUGGAGACA
103 HPV16 25 HR&LR 3043 GUUAGCCUUGAAGUGUAUUUAACUG
104 HPV16 25 HR&LR 3018 UAAUGAAAAGUGGACAUUACAAGAC
105 HPV16 25 HR&LR 2974 GAACUGCAACUAACGUUAGAAACAA
106 HPV16 25 HR&LR 2938 CUGGCUGUAUCAAAGAAUAAAGCAU
107 HPV16 25 HR&LR 2890 GCCAGAGAAAUGGGAUUUAAACAUA
108 HPV16 25 HR&LR 2863 CGCCUAGAAUGUGCUAUUUAUUACA
109 HPV16 25 HR&LR 2828 ACCUACGUGACCAUAUAGACUAUUG
110 HPV16 25 HR&LR 2794 AAAAUACUAACACAUUAUGAAAAUG
111 HPV16 25 HR&LR 2630 UAAUGAGUUUCCAUUUGACGAAAAC
112 HPV16 25 HR&LR 2602 AUAAUAGAUUGGUGGUGUUUACAUU
113 HPV16 25 HR&LR 2555 UACAUCUAACAUUAAUGCUGGUACA
114 HPV16 25 HR&LR 2501 UAUGGAUGUAAAGCAUAGACCAUUG
115 HPV16 25 HR&LR 2444 CUGUUGGAACUACAUAGAUGACAAU
116 HPV16 25 HR&LR 2345 GCAAGGGUCUGUAAUAUGUUUUGUA
117 HPV16 25 HR&LR 2324 UAUGAGUUUAAUGAAAUUUCUGCAA
118 HPV16 25 HR&LR 2282 AUUACUAUAUGGUGCAGCUAACACA
119 HPV16 25 HR&LR 2171 AGGUGAUUGGAAGCAAAUUGUUAUG
120 HPV16 25 HR&LR 2139 AUAAAAUAUAGAUGUGAUAGGGUAG
121 HPV16 25 HR&LR 1957 ACGAUAAUGACAUAGUAGACGAUAG
122 HPV16 25 HR&LR 1914 AAUGAUUGUACAUUUGAAUUAUCAC
123 HPV16 25 HR&LR 1827 UAUAAAACAGGUAUAUCAAAUAUUA
124 HPV16 25 HR&LR 1775 UAUGAUGAUAGAGCCUCCAAAAUUG
125 HPV16 25 HR&LR 1750 AACUAUUAUGUGUGUCUCCAAUGUG
126 HPV16 25 HR&LR 1676 GGGAAUGGUUGUGUUACUAUUAGUA
127 HPV16 25 HR&LR 1584 UUUGGACUUACACCCAGUAUAGCUG
128 HPV16 25 HR&LR 1559 GUGUUGCGAUUGGUGUAUUGCUGCA
129 HPV16 25 HR&LR 1534 GACCAUUUAAAAGUAAUAAAUCAAC
130 HPV16 25 HR&LR 1492 AAUUUAAAGAGUUAUACGGGGUGAG
131 HPV16 25 HR&LR 1417 CUAUAUGCCAAACACCACUUACAAA
132 HPV16 25 HR&LR 1364 UUGCAGUCAGUACAGUAGUGGAAGU
133 HPV16 25 HR&LR 1331 AUGUAGUCAGUAUAGUGGUGGAAGU
134 HPV16 25 HR&LR 1306 AAGGGCGCCAUGAGACUGAAACACC
135 HPV16 25 HR&LR 1238 AUUAUUUGAAAGCGAAGACAGCGGG
136 HPV16 25 HR&LR 1185 CCUAGAUUAAAAGCUAUAUGUAUAG
137 HPV16 25 HR&LR 1150 GUGAUAUUAGUGGAUGUGUAGACAA
138 HPV16 25 HR&LR 1101 UAGAGAUGCAGUACAGGUUCUAAAA
139 HPV16 25 HR&LR 1076 UUACUGCACAGGAAGCAAAACAACA
140 HPV16 25 HR&LR 1029 UAAUGAUUAUUUAACACAGGCAGAA
141 HPV16 25 HR&LR 1004 AUUUGGUAGAUUUUAUAGUAAAUGA
142 HPV16 25 HR&LR 984 UGACAGUGAUACAGGUGAAGAUUUG
143 HPV16 25 HR&LR 848 AGAAACCAUAAUCUACCAUGGCUGA
144 HPV16 25 HR&LR 790 CGUACUUUGGAAGACCUGUUAAUGG
145 HPV16 25 HR&LR 732 UUGUUGCAAGUGUGACUCUACGCUU
146 HPV16 25 HR&LR 702 GGACAGAGCCCAUUACAAUAUUGUA
147 HPV16 25 HR&LR 569 GAGAUACACCUACAUUGCAUGAAUA
148 HPV16 25 HR&LR 524 AGAUCAUCAAGAACACGUAGAGAAA
149 HPV16 25 HR&LR 477 UCCAUAAUAUAAGGGGUCGGUGGAC
150 HPV16 25 HR&LR 412 UAUUAACUGUCAAAAGCCACUGUGU
151 HPV16 25 HR&LR 366 UAGAACAGCAAUACAACAAACCGUU

152 HPV16 25 HR&LR 334 ACAUUAUUGUUAUAGUUUGUAUGGA
153 HPV16 25 HR&LR 306 AGUUUUAUUCUAAAAUUAGUGAGUA
154 HPV16 25 HR&LR 281 UAUGCUGUAUGUGAUAAAUGUUUAA
155 HPV16 25 HR&LR 245 CGGGAUUUAUGCAUAGUAUAUAGAG
156 HPV16 25 HR&LR 209 CAGUUACUGCGACGUGAGGUAUAUG
157 HPV16 25 HR&LR 155 GAGCUGCAAACAACUAUACAUGAUA
158 HPV16 25 HR&LR 130 CAGAAAGUUACCACAGUUAUGCACA
159 HPV16 25 HR&LR 92 AAGAGAACUGCAAUGUUUCAGGACC
160 HPV16 25 HR&LR 57 CCGGUUAGUAUAAAAGCAGACAUUU
161 HPV16 25 HR&LR 18 AUAAAACUAAGGGCGUAACCGAAAU

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 18 consisting essentially of a sequence or a complement thereof selected from the group consisting of. SEQ ID NOs: 163-309 (See Table 2). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 18, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 163-309. In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 18, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of. SEQ ID NOs: 163-299. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 18 comprising SEQ ID NOs: 163-309. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to comprising SEQ ID NO: 163-241,244-274, 276, 277, 279, 280, 282-309.

Table 2: Polyribonucleotide probes for determining HPV 18 nucleic acid.
SEQ ID
NO: Name Sequence 163 HPV18 25 HR&LR -45 7833 UUGGGCAGCACAUACUAUACUUUUC
164 HPV18 25 HR&LR -45 7796 UAAGCUGUGCAUACAUAGUUUAUGC
165 HPV18 25 HR&LR -45 7764 CUGUCUACCCUUAACAUGAACUAUA
166 HPV18 25 HR&LR -45 7738 GUACAACUACUUUCAUGUCCAACAU
167 HPV18 25 HR&LR -45 7658 AUCCACUCCCUAAGUAAUAAAACUG
168 HPV18 25 HR&LR -45 7632 GCUACAACAAUUGCUUGCAUAACUA
169 HPV18 25 HR&LR -45 7561 UUGAACAAUUGGCGCGCCUCUUUGG
170 HPV18 25 HR&LR -45 7536 CUUUUGGGCACUGCUCCUACAUAUU
171 HPV18 25 HR&LR -45 7501 CAAUACAGUACGCUGGCACUAUUGC
172 HPV18 25 HR&LR -45 7476 UGGCUUAUGUCUGUGGUUUUCUGCA
173 HPV18 25 HR&LR(-45)_7423 CCAUUUUAUCCUACAAUCCUCCAUU

174 HPV18 25 HR&LR -45 7398 UAUAAAACUGCACACCUUACAGCAU
175 HPV18 25 HR&LR -45 7370 GGGCUAUAUAUUGUCCUGUAUUUCA
176 HPV18 25 HR&LR -45 7345 GUUUGUGGUAUGGGUGUUGCUUGUU
177 HPV1825 HR&LR(-45)_7320 CCUAGUGAGUAACAACUGUAUUUGU
178 HPV1825 HR&LR(-45)_7291 UUGUGGUUCUGUGUGUUAUGUGGUU
179 HPV18 25 HR&LR -45 7249 GUUACUAUAUUUGUUGGUAUGUGGC
180 HPV18 25 HR&LR -45 7211 CAUUGUAUGGUAUGUAUGGUUGUUG
181 HPV18 25 HR&LR -45 7184 CCUGUGUUUGUGUUUGUUGUAUGAU
182 HPV18 25 HR&LR -45 7123 GUGCCAGGAAGUAAUAUGUGUGUGU
183 HPV18 25 HR&LR -45 7098 AAACCUGCCAAGCGUGUGCGUGUAC
184 HPV18 25 HR&LR -45 7073 UGCUCCAUCUGCCACUACGUCUUCU
185 HPV18 25 HR&LR -45 6982 CUUUAGACUUAGAUCAAUAUCCCCU
186 HPV18 25 HR&LR -45 6911 UGCACCGGCUGAAAAUAAGGAUCCC
187 HPV18 25 HR&LR -45 6876 GUACAAUCUGUUGCUAUUACCUGUC
188 HPV18 25 HR&LR -45 6698 GCAGUAUAGCAGACAUGUUGAGGAA
189 HPV1825 HR&LR(-45)_6672 GGGCAAUAUGAUGCUACCAAAUUUA
190 HPV1825 HR&LR(-45)_6625 CCAGUACCAAUUUAACAAUAUGUGC
191 HPV1825 HR&LR(-45)_6482 GUAUUCUCCCUCUCCAAGUGGCUCU
192 HPV18 25 HR&LR -45 6425 GCCUCAAUCCUUAUAUAUUAAAGGC
193 HPV18 25 HR&LR -45 6254 AGAUACUAAAUGUGAGGUACCAUUG
194 HPV18 25 HR&LR -45 6188 CACAGUUUUGGAAGAUGGUGAUAUG
195 HPV18 25 HR&LR -45 6137 UAAAUCGCGUCCUUUAUCACAGGGC
196 HPV18 25 HR&LR -45 6029 UUCUGAGGACGUUAGGGACAAUGUG
197 HPV18 25 HR&LR -45 6004 GUUCCCAUGCCGCCACGUCUAAUGU
198 HPV18 25 HR&LR -45 5766 GUUCCUGCAGGUGGUGGCAAUAAGC
199 HPV18 25 HR&LR -45 5667 GCAAGAGUUGUAAAUACCGAUGAUU
200 HPV18 25 HR&LR -45 5642 CGUAUAUCUUCCACCUCCUUCUGUG
201 HPV18 25 HR&LR -45 5519 CAGUAUAUUGGUAUACAUGGUACAC
202 HPV1825 HR&LR(-45)_5487 CCAUUGUAUCACCCACGGCCCCUGC
203 HPV1825 HR&LR(-45)_5462 UUACCAUCUACUACCUCUGUAUGGC
204 HPV1825 HR&LR(-45)_5437 UGUAUACACGGGUCCUGAUAUUACA
205 HPV18 25 HR&LR -45 5409 UCCCUUUAACCUCCUCUUGGGAUGU
206 HPV18 25 HR&LR -45 5384 GCCUCUUCCUAUAGUAAUGUAACGG
207 HPV18 25 HR&LR -45 5329 AUCGCGUUCUACUACCUCCUUUGCA
208 HPV18 25 HR&LR -45 5304 ACAUGGACCCUGCAGUGCCUGUACC
209 HPV18 25 HR&LR -45 5249 CAGCCUUUAGUAUCUGCCACGGAGG
210 HPV18 25 HR&LR -45 5224 ACCUUCCCCAGAAUAUAUUGAACUG
211 HPV18 25 HR&LR -45 5160 UUACCCGCAGCGGUACACAAAUAGG
212 HPV18 25 HR&LR -45 5118 GGACUGUUCGCUUUAGUAGAUUAGG
213 HPV18 25 HR&LR -45 5021 GACACUACAUUAACAUUUGAUCCUC
214 HPV18 25 HR&LR -45 4971 CACGUCCAUCCUCUUUAAUUACAUA
215 HPV1825 HR&LR(-45)_4946 UCAGUGGCUAACCCUGAGUUUCUUA
216 HPV1825 HR&LR(-45)_4833 UACAAACAUUUGCUUCUUCUGGUAC
217 HPV18 25 HR&LR -45 4737 CGUCCAUUAUUGAAGUUCCACAAAC
218 HPV18 25 HR&LR -45 4701 CCACAACCAAUUUUACCAAUCCUGC
219 HPV18 25 HR&LR -45 4676 CCUUCGUCUACCUCUGUGUCUAUUU
220 HPV18 25 HR&LR -45 4634 ACAUCUGCGGGUACAACUACACCUG
221 HPV18 25 HR&LR -45 4591 UGCACCUAGGCCUACGUUUACUGGC
222 HPV18 25 HR&LR -45 4566 AGGACUCCAGUGUGGUUACAUCAGG
223 HPV18 25 HR&LR -45 4483 AGUGGUGGAUGUUGGUCCUACACGU
224 HPV18 25 HR&LR -45 4455 ACAUUCCAUUGGGUGGGCGUUCCAA

225 HPV18 25 HR&LR -45 4375 AUUGCAAUGGUCAAGCCUUGGUAUA
226 HPV18 25 HR&LR -45 4276 GGCUUCGGUAACUGACUUAUAUAAA
227 HPV18 25 HR&LR -45 4234 UAAUAAAAGUAUGGUAUCCCACCGU
228 HPV1825 HR&LR(-45)_4113 CCCAUGUUACUAUUGCAUAUACAUG
229 HPV1825 HR&LR(-45)_4072 CUGCCACAGCAUUCACAGUAUAUGU
230 HPV18 25 HR&LR -45 4047 GUGUAUAUUGUGGUAAUAACGUCCC
231 HPV18 25 HR&LR -45 3971 AUGCAUGUAUGUGUGCUGCCAUGUC
232 HPV18 25 HR&LR -45 3922 GCUGUAGUACCAAUAUGUUAUCACU
233 HPV18 25 HR&LR -45 3888 AUAUUGGUGGGAUACAUGACAAUGU
234 HPV18 25 HR&LR -45 3863 UGUUGCAAUUCCAGAUAGUGUACAA
235 HPV18 25 HR&LR -45 3823 CAUACCAUAGUGAAACACAAAGAAC
236 HPV18 25 HR&LR -45 3752 CUAUAGAGAUAUAUCAUCCACCUGG
237 HPV18 25 HR&LR -45 3727 ACAGAUUGCGAAAACAUAGCGACCA
238 HPV18 25 HR&LR -45 3647 AAGACGGAAACUCUGUAGUGGUAAC
239 HPV18 25 HR&LR -45 3622 CAGCUACACCUACAGGCAACAACAA
240 HPV1825 HR&LR(-45)_3597 GGACCUGUCAACCCACUUCUCGGUG
241 HPV1825 HR&LR(-45)_3572 UGGACUCGCGGAGAAGCAGCAUUGU
242 HPV1825 HR&LR(-45)_3547 CGGCUGCUACACGACCUGGACACUG
243 HPV18 25 HR&LR -45 3499 AUUCCAGCACCGUGUCCGUGGGCAC
244 HPV18 25 HR&LR -45 3454 CCGCUACUCAGCUUGUUAAACAGCU
245 HPV18 25 HR&LR -45 3382 GGGAAGUACAUUUUGGGAAUAAUGU
246 HPV18 25 HR&LR -45 3315 GAAGGGUACAACACGUUUUAUAUAG
247 HPV18 25 HR&LR -45 3269 CAAAACCGCUACCUGUGUAAGUCAC
248 HPV18 25 HR&LR -45 3244 AUAUGACUGAUGCAGGAACAUGGGA
249 HPV18 25 HR&LR -45 3219 UAUGUAGCAUGGGACAGUGUGUAUU
250 HPV18 25 HR&LR -45 3168 GGCCAAACAGUACAAGUAUAUUUUG
251 HPV18 25 HR&LR -45 3134 GAAUACAGAACCUACUCACUGCUUU
252 HPV18 25 HR&LR -45 3080 AAGUCGAUACAAAACCGAGGAUUGG
253 HPV1825H R&LR(-45)_2972 ACAUGGCAUACAGACAUUAAACCAC
254 HPV1825 HR&LR(-45)_2938 GUUGGGAAAAUGCAAUAUUCUUUGC
255 HPV1825 HR&LR(-45)_2903 CAUAGACAGCCAAAUACAGUAUUGG
256 HPV18 25 HR&LR -45 2645 GCAAAGGAUAAUAGAUGGCCAUAUU
257 HPV18 25 HR&LR -45 2612 CCUCCAAUACUACUAACCACAAAUA
258 HPV18 25 HR&LR -45 2527 CUUUGAUACCUAUAUGAGAAAUGCG
259 HPV18 25 HR&LR -45 2475 CAGAUACUAAGGUGGCCAUGUUAGA
260 HPV18 25 HR&LR -45 2270 CUGCGAUACCAACAAAUAGAGUUUA
261 HPV18 25 HR&LR -45 2202 CACAGUGGAUACGAUUUAGAUGUUC
262 HPV18 25 HR&LR -45 2065 UGAAUAUGCCUUAUUAGCAGACAGC
263 HPV18 25 HR&LR -45 2036 GAGCUGACAGAUGAAAGCGAUAUGG
264 HPV18 25 HR&LR -45 1944 CUGAGUGGAUACAAAGACUUACUAU
265 HPV18 25 HR&LR -45 1918 UAUUAGUGAAGUAAUGGGAGACACA
266 HPV1825 HR&LR(-45)_1829 CACGUACCUGAAACUUGUAUGUUAA
267 HPV1825 HR&LR(-45)_1802 GUUGCUAAAGGUUUAAGUACGUUGU
268 HPV18 25 HR&LR -45 1777 CAAAUGUGGUAAGAGUAGACUAACA
269 HPV18 25 HR&LR -45 1751 GUAUUAAUAUUAGCCCUGUUGCGUU
270 HPV18 25 HR&LR -45 1726 UCAAUGUCUAGACUGUAAAUGGGGA
271 HPV18 25 HR&LR -45 1572 ACACAUAUGGGCUAUCAUUUACAGA
272 HPV18 25 HR&LR -45 1536 ACAAUAAACAAGGAGCUAUGUUAGC
273 HPV18 25 HR&LR -45 1493 CCACAAUGUACCAUAGCACAAUUAA
274 HPV18 25 HR&LR -45 1455 ACGGUACAAGUGACAAUAGCAAUAU
275 HPV18 25 HR&LR -45 1429 CACAGAGGGCAACAACAGCAGUGUA

276 HPV18 25 HR&LR -45 1399 CGGCAGUACGGAGGCUAUAGACAAC
277 HPV18 25 HR&LR -45 1360 AACUACAAAUGGCGAACAUGGCGGC
278 HPV18 25 HR&LR -45 1216 GCGGCUGGAGGUGGAUACAGAGUUA
279 HPV1825 HR&LR(-45)_1149 CACAAGUGUUGCAUGUUUUAAAACG
280 HPV1825 HR&LR(-45)_1072 ACAAGGAACAUUUUGUGAACAGGCA
281 HPV18 25 HR&LR -45 959 GGCUGGUUUUAUGUACAAGCUAUUG
282 HPV18 25 HR&LR -45 885 CGUGGUGUGCAUCCCAGCAGUAAGC
283 HPV18 25 HR&LR -45 857 UUUCUGAACACCCUGUCCUUUGUGU
284 HPV18 25 HR&LR -45 816 UAGAAAGCUCAGCAGACGACCUUCG
285 HPV18 25 HR&LR -45 791 UGUGAAGCCAGAAUUGAGCUAGUAG
286 HPV18 25 HR&LR -45 695 GAAGAAAACGAUGAAAUAGAUGGAG
287 HPV18 25 HR&LR -45 670 UCACGAGCAAUUAAGCGACUCAGAG
288 HPV18 25 HR&LR -45 645 AUGAAAUUCCGGUUGACCUUCUAUG
289 HPV18 25 HR&LR -45 620 AUUGUAUUGCAUUUAGAGCCCCAAA
290 HPV18 25 HR&LR -45 589 UAUGCAUGGACCUAAGGCAACAUUG
291 HPV1825 HR&LR(-45)_554 CCAACGACGCAGAGAAACACAAGUA
292 HPV1825 HR&LR(-45)_529 GCAACCGAGCACGACAGGAACGACU
293 HPV1825_H R&LR(-45)_489 AACAUAGCUGGGCACUAUAGAGGCC
294 HPV18 25 HR&LR -45 344 UUAUUCAGACUCUGUGUAUGGAGAC
295 HPV18 25 HR&LR -45 264 GUGGUGUAUAGAGACAGUAUACCCC
296 HPV18 25 HR&LR -45 216 GUAUUGGAACUUACAGAGGUAUUUG
297 HPV18 25 HR&LR -45 179 GCAAGACAUAGAAAUAACCUGUGUA
298 HPV18 25 HR&LR -45 154 UGUGCACGGAACUGAACACUUCACU
299 HPV18 25 HR&LR -45 92 ACACCACAAUACUAUGGCGCGCUUU

304 HPV18_2877 GACCACUAUGAAAAUGACAGUAAAG
305 HPV18_1298 CUGUUUACAAUAUCAGAUAGUGGCU
306 HPV18_1241 AGUCCACGGUUACAAGAAAUAUCUU

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 45 consisting essentially of a sequence or a complement thereof selected from the group consisting of. SEQ ID NOs: 842-974 (See Table3). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 45, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 842-974. In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 45, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of. SEQ ID NOs: 842-968. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 45 comprising SEQ ID NOs: 842-974. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to comprising SEQ ID NO: 842-849, 851-893, 895-917, 919-929, 931, 933-936, 938-974.

Table 3: Polyribonucleotide probes for determining HPV 45 nucleic acid.
SEQ ID
NO: Name Sequence 842 HPV45 25 HR&LR -18 7834 GGCCCUAUAACACAUACCUUUUCUU
843 HPV45 25 HR&LR -18 7754 CCAACAAUCUGUCUACUUGUUACAU
844 HPV45 25 HR&LR -18 7726 UAAUUGGCGUGUAGAACCACUUUCU
845 HPV45 25 HR&LR -18 7646 GCACAACUGUAUCCACACCCUAUGU
846 HPV45 25 HR&LR -18 7552 ACAUAGUUUAACCUACUGGCGCGCC
847 HPV45 25 HR&LR -18 7527 CUAAACUGGCACAUUUACAACCCCU
848 HPV45 25 HR&LR -18 7495 GUGGCUUAUAUGUGACCUUUUAAAC
849 HPV45 25 HR&LR -18 7440 GCAUCCAUUUUACUUAUAAUCCUCC
850 HPV45 25 HR&LR -18 7385 CUUUGUACCCUAUAUUCUUUCCUGU
851 HPV45 25 HR&LR -18 7322 UAAUAGUGUUGUGUAGGGUUGCACC
852 HPV45 25 HR&LR -18 7282 GGUGUUACUGUACAUAAUUGUGGUA
853 HPV45_25 HR&LR(-18)_7250 GUGUAUGUAUGAAUGUGCCUUGUGG
854 HPV45_25 HR&LR(-18)_7225 UACUGUAUUUUGUUUGUUUGCGUGC
855 HPV45 25 HR&LR -18 7106 CAUCUAGGCCUGCCAAACGUGUACG
856 HPV45 25 HR&LR -18 7081 GCUUCCACGUCUACUGCAUCUACUG
857 HPV45 25 HR&LR -18 7052 CUACCAUAGGACCUCGUAAGCGUCC
858 HPV45 25 HR&LR -18 7027 GUUCAGGCUGGGUUACGUCGUAGGC
859 HPV45 25 HR&LR -18 6911 AUACUACACCUCCAGAAAAGCAGGA
860 HPV45 25 HR&LR -18 6885 AUCAGUUGCUGUUACCUGUCAAAAG
861 HPV45 25 HR&LR -18 6697 UUUAAGCAGUAUAGUAGACAUGUGG
862 HPV45 25 HR&LR -18 6672 GCCAAGUACAUAUGACCCUACUAAG
863 HPV45 25 HR&LR -18 6505 GGCUCUAUUAUUACUUCUGAUUCUC
864 HPV45 25 HR&LR -18 6479 GUUGUGUGUAUUCCCCUUCUCCCAG
865 HPV45 25 HR&LR -18 6454 GCUAAUAUGCGUGAAACCCCUGGCA
866 HPV4525_H R&LR(-18)_6426 UACGGACCUAUAUAUUAAAGGCACU
867 HPV45_25 HR&LR(-18)_6272 CAUUAGACAUUUGUCAAUCCAUCUG
868 HPV45 25 HR&LR -18 6247 UUGCAGGAUACAAAGUGCGAGGUUC
869 HPV45 25 HR&LR -18 6142 GCACAAUUGCAACCUGGUGACUGUC
870 HPV45 25 HR&LR -18 6018 AGCUGUUAUUACGCAGGAUGUUAGG
871 HPV45 25 HR&LR -18 5833 GUAGCUUUACCCGAUCCUAAUAAAU
872 HPV45 25 HR&LR -18 5791 GCUGUUCCUAAGGUAUCCGCAUAUC
873 HPV45 25 HR&LR -18 5766 ACCUAAUGGUGCAGGUAAUAAACAG
874 HPV45 25 HR&LR -18 5741 UAGGCAAUCCAUAUUUUAGGGUUGU
875 HPV45 25 HR&LR -18 5654 CUUCUGUGGCCAGAGUUGUCAGCAC
876 HPV45 25 HR&LR -18 5534 CACACAAUAUUAUUUAUGGCCAUGG
877 HPV45 25 HR&LR -18 5490 UCUCCUACCAAUGCUUCCACCACCA
878 HPV45_25 HR&LR(-18)_5465 CCAUACUCCUAUGUGGCCUAGUACA
879 HPV45_25 HR&LR(-18)_5437 AUACUGGCCCGGACAUUAUAUUGCC
880 HPV4525 HR&LR(-18)_5402 AGUACCAUUAACAUCUGCAUGGGAU
881 HPV45 25 HR&LR -18 5372 UACUGCUGCAUCCUCUUACAGUAAU
882 HPV45 25 HR&LR -18 5347 CAAAGUAUUCCUUGACCAUGCCUUC
883 HPV45 25 HR&LR(-18)_5314 CACCUAGCACUAUACACAAAUCAUU

884 HPV45 25 HR&LR -18 5289 GACUUCCCACCUCCUGCGUCCACUA
885 HPV45 25 HR&LR -18 5254 CUACAAAUGAUAGUGACCUGUUUGA
886 HPV45 25 HR&LR -18 5209 CCAUUGCUGCUACAGAGGAAAUUGA
887 HPV45_25_HR&LR(-18)_5111 CACUGUUAGAUUUAGUAGAUUGGGU
888 HPV45_25_HR&LR(-18)_5038 CCAGUAAUGUUCCUGAUUCCGAUUU
889 HPV45 25 HR&LR -18 5013 GACACCACACUAUCCUUUGAGCCUA
890 HPV45 25 HR&LR -18 4974 UCGUUGGUUACAUUUGAUAAUCCAG
891 HPV45 25 HR&LR -18 4926 AAUCAACAGGUCCGUGUGUCCACCU
892 HPV45 25 HR&LR -18 4837 CAUCUUCUGGGUCAGGUACGGAACC
893 HPV45 25 HR&LR -18 4781 UGGUACACCAACAUCGGGCAGCCAU
894 HPV45 25 HR&LR -18 4716 GCAUUUUCUGAUCCCUCUAUUAUUG
895 HPV45 25 HR&LR -18 4679 CUCUGUUUCUAUUUCGUCAACUAGU
896 HPV45 25 HR&LR -18 4654 UGUUGGACAUCACACCUACCGUGGA
897 HPV45 25 HR&LR -18 4573 UUGCCUCUGGUGCUCCGGUUCCCAC
898 HPV45 25 HR&LR -18 4463 CAGGUCUAAUACUGUUGUGGAUGUU
899 HPV45_25 HR&LR(-18)_4367 UUUACAGUGGUCUAGCCUUGGGAUA
900 HPV45_25 HR&LR(-18)_4224 GUUUAAUAAACCAUGGUAUCCCACC
901 HPV45_25 HR&LR(-18)_4158 AUACCUGUGAUGUGCAUGUUGUUGU
902 HPV45 25 HR&LR -18 4106 GCAUGCUUUACACACCAUACAAUAA
903 HPV45 25 HR&LR -18 4053 GCAUUUGCUGUAUACAUUUGUUGCU
904 HPV45 25 HR&LR -18 3989 UGUGUGUGCUUUUGCUUGGUUGUUG
905 HPV45 25 HR&LR -18 3944 GUGCCUUUAUGUGUGCUGCAAUGUC
906 HPV45 25 HR&LR -18 3857 GGGAUACAUGACUAUAUGAAUCUGU
907 HPV45 25 HR&LR -18 3832 UUCCUAACAGUGUACAAAUCUCGGU
908 HPV45 25 HR&LR -18 3717 UACUCAGAAAUAUCCUCCACCUGGC
909 HPV45 25 HR&LR -18 3685 UAAGAUAUAGGCUACGCAAAUAUGC
910 HPV45 25 HR&LR -18 3612 AGAAGGAAAGUGUGUAGUGGUAACA
911 HPV45 25 HR&LR -18 3585 CUGUGUUCAAGUACAAGUAACAACA
912 HPV4525_H R&LR(-18)_3535 UCACAGAGCAGCACCACGGACGUGU
913 HPV4525 HR&LR(-18)_3492 CACAUCCAGACGCCGGCUACUAAGC
914 HPV4525 HR&LR(-18)_3429 AGACAGCUACAACACGCCUCCACGU
915 HPV45 25 HR&LR -18 3325 GAAAUAGUAAUACGUGGGAAGUACA
916 HPV45 25 HR&LR -18 3241 GUGUUAGCUAUUGGGGUGUAUAUUA
917 HPV45 25 HR&LR -18 3216 GGGAUAUGGGACAAAACAGCAGCAU
918 HPV45 25 HR&LR -18 3173 GAACUAUGUAGUAUGGGACAGUAUA
919 HPV45 25 HR&LR -18 3134 CGUGCACGUAUACUUUGAUGGCAAC
920 HPV45 25 HR&LR -18 3092 GAAUACAGAACCGUCGCAGUGUUUU
921 HPV45 25 HR&LR -18 3039 AGCAAGUAUAACAAUGAGGAAUGGA
922 HPV45 25 HR&LR -18 2918 UACAGCAAGGGAACAUGGUAUUACC
923 HPV45 25 HR&LR -18 2883 UGGCAACUUAUACGUUUGGAAAAUG
924 HPV45 25 HR&LR -18 2850 GACAGUAAAGACAUAAACAGCCAAA
925 HPV4525_H R&LR(-18)_2765 GACGAUGAAGAUGCAGACACCGAAG
926 HPV4525_H R&LR(-18)_2642 ACGGUAUUUACAUUUCCACAUGCAU
927 HPV45 25 HR&LR -18 2586 CAUCCAAUAUUGAUCCAGCAAAAGA
928 HPV45 25 HR&LR -18 2560 GCUAAAAUGUCCUCCAAUCCUAUUA
929 HPV45 25 HR&LR -18 2431 AGCAGAUACUAAGGUAGCCAUGUUG
930 HPV45 25 HR&LR -18 2358 GUUUUAUACAUUUCCUACAAGGUGC
931 HPV45 25 HR&LR -18 2266 GGCACUAAAGGAAUUUCUUAAAGGA
932 HPV45 25 HR&LR -18 1781 UUGUUGCACGUACCUGAAACAUGUA
933 HPV45 25 HR&LR -18 1754 CUAACUGUUGCAAAAGGCUUAAGCA
934 HPV45 25 HR&LR -18 1676 GCCCAUAUCCAAUGUUUAGAUUGUA

935 HPV45 25 HR&LR -18 1599 GGGUAAUGGCUAUAUUUGGAGUUAA
936 HPV45 25 HR&LR -18 1541 CUGUCAUUUACGGAUUUGGUUAGAA
937 HPV45 25 HR&LR -18 1516 GGCAGUAUUUAAAGACAUAUAUGGG
938 HPV4525_HR&LR(-18)_1474 AAAGGAGCUAUUACAAGCAAGUAAC
939 HPV4525_HR&LR(-18)_1449 AUCCGCAUUGCAGUAUUACAGAACU
940 HPV45 25 HR&LR -18 1424 AGUAGUGACAAUGCAGAAAAUGUAG
941 HPV45 25 HR&LR -18 1399 UAGUACACAAAGUAGUGGUGGGGAU
942 HPV45 25 HR&LR -18 1365 UAAACACUAAUGCGGAAAAUGGCGG
943 HPV45 25 HR&LR -18 1338 UGGAAGCUGCAGAGACUCAGGUAAC
944 HPV45 25 HR&LR -18 1242 GUCCACGGUUACAAGAAAUUUCAUU
945 HPV45 25 HR&LR -18 1217 CAGCUAAGUGUGGAUACGGAUCUAA
946 HPV45 25 HR&LR -18)_1 153 GGUGUUGCAUCUUUUAAAACGAAAG
947 HPV45 25 HR&LR -18 1124 CAUGCGCAGGAAGUUCAGAAUGAUG
948 HPV45 25 HR&LR -18 1072 ACAAUUAUCCAUUUGUGAACAGGCA
949 HPV45 25 HR&LR -18 954 GUAAUGGCUGGUUCUUUGUAGAAAC
950 HPV45_25 HR&LR(-18)_897 CUAACCAAUAAUCUACAAUGGCGGA
951 HPV45_25 HR&LR(-18)_832 GGACCUUAGAACACUACAGCAGCUG
952 HPV45_25 HR&LR(-18)_799 CAGAAUUGAGCUUACAGUAGAGAGC
953 HPV45 25 HR&LR -18 649 AGAUCCUGUUGACCUGUUGUGUUAC
954 HPV45 25 HR&LR -18 624 UGCAUUUGGAACCUCAGAAUGAAUU
955 HPV45 25 HR&LR -18 596 CCCCGGGAAACACUGCAAGAAAUUG
956 HPV45 25 HR&LR -18 570 CAAGUAUAGCAAUAAGUAUGCAUGG
957 HPV45 25 HR&LR -18 536 ACGGCAAGAAAGACUUCGCAGACGU
958 HPV45 25 HR&LR -18 511 AGUGUAAUACAUGUUGUGACCAGGC
959 HPV45 25 HR&LR -18 486 AGCAUAGCUGGACAGUACCGAGGGC
960 HPV45 25 HR&LR -18 461 CCUUAAGGACAAACGAAGAUUUCAC
961 HPV45 25 HR&LR -18 348 AACUCUGUAUAUGGAGAGACACUGG
962 HPV45 25 HR&LR -18 265 UGUAUAGAGACUGUAUAGCAUAUGC
963 HPV4525 HR&LR(-18)_218 GGAACGCACAGAGGUAUAUCAAUUU
964 HPV45_25 HR&LR(-18)_188 UAUUGCCUGUGUAUAUUGCAAAGCA
965 HPV4525 HR&LR(-18)_163 UGAAUACAUCACUACAAGACGUAUC
966 HPV45 25 HR&LR -18 138 AAGCUACCAGAUUUGUGCACAGAAU
967 HPV45 25 HR&LR -18 113 UGACGAUCCAAAGCAACGACCCUAC
968 HPV45 25 HR&LR -18 87 AAAGUGCAUUACAGGAUGGCGCGCU

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 31 consisting essentially of a sequence or a complement thereof selected from the group consisting of. SEQ ID NOs: 310-454 (See Table4). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 31, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 310-454. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 31 comprising SEQ
ID NOs: 310-454.

Table 4: Polyribonucleotide probes for determining HPV 31 nucleic acid.
SEQ ID
NO: Name Sequence 321 HPV31_7396 UAGUAAAAGUUGUACACCCGGUCCG

323 HPV31_7325 UGUUCCUACUUGUUCCUGCUCCUCC

334 HPV31_6424 AUACUUUCCUACACCUAGCGGCUCC

336 HPV31_6358 UGAAUCGGUCCCUACUGACUUAUAU

357 HPV31_5046 CACUGUUAGAUAUAGUAGACUAGGU
358 HPV31_4990 CCCGACUUUCUAGAUAUUAUAGCAU

382 HPV31_3789 CAACAGGAUAUAUGACUAUUUAGCC
383 HPV31_3673 UUGGACAUGUACAGAUGGAAAACAU
384 HPV31_3645 UGUAUGAACAAGUGUCAUCUACAUG

395 HPV31_3073 CACCAUGCAUUAUACUAACUGGAAA

408 HPV31_2222 UAAUACAUGGUGCACCUAAUACAGG
409 HPV31_2109 AGGUGACUGGAGGGACAUAGUAAAG

420 HPV31_1408 GGUAAAGCUGCUAUGUUAGGUAAAU
421 HPV31_1369 CCAACACGUAAUAUAUUGCAAGUGU
422 HPV31_1344 ACAUAGUGAACGAGAGAAUGAAACU

433 HPV31_848 AGACUGUAACUACAAUGGCUGAUCC
434 HPV31_814 CUCAUUUGGAAUCGUGUGCCCCAAC
435 HPV31_789 GCAUAUUGCAAGAGCUGUUAAUGGG

447 HPV31_287 CGGAGUGUGUACAAAAUGUUUAAGA

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 33 consisting essentially of a sequence or a complement thereof selected from the group consisting of. SEQ ID NOs: 455-579 (See Table 5). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 33, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 455-579. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 33 comprising SEQ
ID NOs:455-579.

Table 5: Polyribonucleotide probes for determining HPV 33 nucleic acid.
SEQ ID
NO: Name Sequence 477 HPV33_6630 CACAAGUAACUAGUGACAGUACAUA

503 HPV33_4993 CCUGAAGACACAUUACAAUUUCAAC

516 HPV33_4435 ACUGACCCACCUACAGCUGCAAUCC

518 HPV33_4119 CAUGGUGGUGUUUUAACAUUGUUGU

543 HPV33_2454 GAUGAUUACAUGAGAAAUGCGUUAG

554 HPV33_1426 CGUUGCAGGAAAUUAGUAAUGUUCU
555 HPV33_1395 GAGACAAAUGUAGAUAGCUGUGAAA
556 HPV33_1345 UAAAUGACUUAGAAUCUAGUGGGGU

567 HPV33_742 GU UGUCACACUUGUAACACCACAGU
568 HPV33_717 ACAGCUGAUUACUACAUUGUAACCU
569 HPV33_617 AUAUCCUGAACCAACUGACCUAUAC

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 35 consisting essentially of a sequence or a complement thereof selected from the group consisting of. SEQ ID NOs: 580-722 (See Table 6). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 35, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ

ID NOs:580-722. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 35 comprising SEQ
ID NOs:580-722.

Table 6: Polyribonucleotide probes for determining HPV 35 nucleic acid.
SEQ ID
NO: Name Sequence 618 HPV35_5440 UAUUACUAACUCUGUACUACCGGUA

625 HPV35_5120 AGUGGAAAAGCUAUAGGGGCACGGG
626 HPV35_5094 GUAAUAAACGUACUAUGCAUACACG

637 HPV35_4605 CUACAGAUACCACACCUGCUAUUUU

639 HPV35_4553 CCUGUUGUUACACCAAGGGUCCCAC

651 HPV35_3933 ACUGUGGGUUACUGUAGCAACACCA
652 HPV35_3889 CUAUCUGUGUCAUUAUACUCAGCAU

663 HPV35_3399 AGAAGACAAAUCACAAACGACUUCG

676 HPV35_2679 AGAGGUCAAAGAAAAUGAUGGAGAC

688 HPV35_1744 CGUAGUACCCCAGCUGCGUUAUAUU
689 HPV35_1698 GCUAUGUAUUUCAGCUGCAAGUAUG
690 HPV35_1619 GGGCUAUGGUAAUUCUAGCAUUAUU

701 H PV35_1051 GAAACAGAGACAGCACAAGCAUUAU
702 HPV35_970 GACGAAAAUGAAGAUGACUGUGACA
703 HPV35_945 UAGACGUACGGGAUCCAGUGUAGAG

715 HPV35_284 CCAUAUGGAGUAUGCAUGAAAUGUU

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 39 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs: 723-841 (See Table 7). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 39, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 723-841. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 39 comprising SEQ
ID NOs: 723-841.

Table 7: Polyribonucleotide probes for determining HPV 39 nucleic acid.
SEQ ID
NO: Name Sequence 732 HPV39_7273 AUGACAGUUUCAUGUGUGAUUGCAC
733 HPV39_7203 CCUUAUGUGUUGAGUGUAUAUGUGU
734 HPV39_7173 CCUUGUUAUGUGUGUGUAUGUUGUU

759 HPV39_5543 ACAACAUAUGCAAUAACCAUUCAGG

771 HPV39_5178 GCACACAAAUUGGAGCGCAAGUACA
772 HPV39_5121 AAGGAACAGUAAGGUUUAGUAGGCU

785 HPV39_4333 ACCAGACGUUGUUGAUAAAGUUGAG
786 HPV39_4297 CCUAUAUAGAACCUGUAAACAAUCG

797 HPV39_3591 UGGACCAUCUUAACAACCCACUCCA

822 HPV39_1568 UAUCCUUUACUGACCUGGUACGUAC
823 HPV39_1531 GCUGCAAUGCUAACACAAUUUAAAG
824 HPV39_1478 CCAAAUCUCCAACUGCACAAAUUAA

835 HPV39_567 GAGAAACCCAAGUAUAACAUCAGAU
836 HPV39_464 CACCUAAAUAGCAAACGAAGAUUUC

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 51 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs 975-1120: (See Table 8). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 51, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 975-1120. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 51 comprising SEQ
ID NOs: 975-1120.

Table 8: Polyribonucleotide probes for determining HPV 51 nucleic acid.
SEQ ID
NO: Name Sequence 982 HPV51_7485 UAGUGCAUACAUCCGCCCGCCCACG
983 HPV51_7427 AAGUUUUAAACCACAACUGCCAGUU

994 HPV51_6707 CUACCAUUCUUGAACAGUGGAAUUU

996 HPV51_6597 CUUUAAGCAAUAUAUUAGGCAUGGG

1007 HPV51_5897 AUGACACAGAAAAUUCACGCAUAGC
1008 HPV51_5773 CCGGAUCCAAAUUUAUAUAAUCCAG

1021 HPV51_5247 CACUCCUCUUUGUCUAGGCAGUUGC

1038 HPV51_4425 CACCAUACUGAACCUUCUAUAGUAA

1040 HPV51_4373 AGGCGUGGUGGAUAUUGCUCCUGCA

1052 HPV51_3689 GGCAUUGUUACCAUUGUGUUUGACA

1065 HPV51_3151 GUGGGUAAAGACAAAUGGAAAUGUG

1077 HPV51_2363 AGCCACUAGAGGAUGCUAAAAUAGC
1078 HPV51_2307 GUUUAUGCAAGGGUCCAUUAUUUCA

1089 HPV51_1559 UUUCCCCAAUGGUAGCAGAAAAUUU
1090 HPV51_1534 GAUUGGGUUUGUGCAUUGUUUGGCG
1091 HPV51_1489 AAUGAGUUGGUACGGGUGUUUAAAA

1102 HPV51_982 GAAAAUGCAGAUGAUACAGGAUCUG
1103 HPV51_957 AGAUAAUGUUUCGGAUGAUGAGGAU
1104 HPV51_862 CUAGCAACGGCGAUGGACUGUGAAG

1116 HPV51_323 AUAGACGUUAUAGCAGGUCUGUGUA

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 52 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs: 1121-1252 (See Table 9). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 52, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 1121-1252. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 52 comprising SEQ ID NOs:
1121-1252.

Table 9: Polyribonucleotide probes for determining HPV 52 nucleic acid.
SEQ ID
NO: Name Sequence 1128 HPV52_7540 CCAUUUUAAAUCCUAACCGAAUUCG

1139 HPV52_7143 GUUAAAAGGUAACCAUUGUCUGUUG
1140 HPV52_7112 GGCCCCACGUACCUCCACAAAGAAG

1152 HPV52_6275 GGAUUUUAAUACCUUGCAAGCUAGU

1160 HPV52_5435 GGUAUUGACUUUGUAUAUCAACCCA
1161 HPV52_5385 CUUCCACACUUUCUACCCAUAAUAA

1172 HPV52_4859 ACAUUUGUUACCUCUACUGACAGCA

1187 HPV52_4157 AUAACUGUACAUGUAGAUUGGCUAC

1198 H PV52_3509 UGCGGGGACAACAAUCCGUGGACAG

1211 HPV52_2847 GACUCGAAUGGAAUGUGUUUUGUUU

1223 HPV52_1723 CCAAACUAAUGUCACAGCUGUUAAA
1224 HPV52_1670 GCUUAUACUGCUGCUAAUUAGGUUU
1225 HPV52_1585 CAUCAGUUGCAGAAGGAUUAAAAGU

1236 HPV52_853 ACAACCCUGCAAUGGAGGACCCUGA

1249 HPV52_145 UGUGUGAGGUGCUGGAAGAAUCGGU
1250 H PV52_120 ACACGACCCCGGACCCUGCACGAAU

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 56 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs: 1253-1367 (See Table
10). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 56, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 1253-1367. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 56 comprising SEQ ID NOs:
1253-1367.

Table 10: Polyribonucleotide probes for determining HPV 56 nucleic acid.
SEQ ID
NO: Name Sequence 1270 HPV56_6727 CAAAAUUACUUUGUCUGCAGAGGUU

1283 HPV56_5750 GUGACUAAGGACAAUACCAAAACAA
1284 HPV56_5524 UCCUCCUUUGCAUUAUGGCCUGUGU

1293 HPV56_4885 GCAGCUCCUAGAUUAUAUAGAAAAG

1305 HPV56_4045 CCUCUGUGUUUUCCAGUUGUAUAUU

1307 HPV56_3993 UGCUUUUGUGUUUGUUUGCUUGUGU

1319 HPV56_3247 CUACACAGACUUUGAACAAGAGGCC
1320 HPV56_3197 GGGGUAGACUAUAGAGGUAUAUAUU

1331 H PV56_2124 CAGUGGAUAAAGCACAUAUGUAGUA
1332 HPV56_1957 AAGUAACAGAUGAUAGCCAAAUUGC

1344 HPV56_1170 CCAUUAAGGGAUAUUAGUAAUCAGC
1345 H PV56_1108 UACAAACAGCACAUGCAGAUAAACA

1356 HPV56_529 GGAGACAAACAUCUAGAGAACCUAG
1357 HPV56_504 UGGACCGGGUCAUGUUUGGGGUGCU
1358 HPV56_479 ACGAUUUCAUCUAAUAGCACAUGGU

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 58 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs: 1368-1497 (See Table
11 ). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 58, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of. SEQ
ID NOs: 1368-1497. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 58 comprising SEQ ID NOs:
1368-1497.

Table 11: Polyribonucleotide probes for determining HPV 58 nucleic acid.
SEQ ID
NO: Name Sequence 1375 HPV58_7384 CUGCCUAUUAUGCAUACCUAUGUAA
1376 HPV58_7359 UGUCCCUAAAUUGCCCUACCCUGCC

1388 HPV58_6453 GUCCCGGAUGACCUUUAUAUUAAAG

1401 HPV58_5423 GUGUCCAUACCAUUAAAUACUGGAU

1413 HPV58_4929 GUCGCAACACCCAACAAGUUAAGGU

1427 HPV58_4139 CACAUGGUGGUAUGGUAUUGUAAAU

1438 HPV58_3487 GUAUACAGACUGCGCCGUGGACAGU

1440 HPV58_3437 CGACGACUCGAUUUACCAGACUCCA

1451 HPV58_2873 GCUAUAAUGUAUACAGCCAGACAAA

1453 HPV58_2794 AAUCCUAGACAUAUACGAAGCUGAU

1464 HPV58_1800 AAGUCAAGCAUGUGCCUUAUAUUGG
1465 HPV58_1770 AUGUAUGAUUAUCGAGCCACCAAAA

1477 HPV58_1005 CGAUAGUGGUACAGAUUUAAUAGAG
1478 HPV58_958 CGAAGAACAGGAGAUAAUAUUUCAG

1489 HPV58_533 CCCCGACGUAGACAAACACAAGUGU

1491 HPV58_360 AUGGAGACACAUUAGAACAAACACU

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 59 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs: 1498-1646 (See Table
12). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 59, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 1498-1646. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 59 comprising SEQ ID NOs:
1498-1646.

Table 12: Polyribonucleotide probes for determining HPV 59 nucleic acid.
SEQ ID
NO: Name Sequence 1499 HPV59_7735 ACUACUGUGCAAUCCAAGAAUGUGU

1508 HPV59_7367 AGGUGUGUUUGUUCCUUCAUUUUGU

1522 HPV59_6663 CUAAUGUAUACACACCUACCAGUUU

1533 HPV59_5857 CCUUCCAGAUAACACAGUAUAUGAU

1559 HPV59_4685 GUAGCUCUAGUUUUAUAAAUCCUGC

1585 HPV59_3354 ACCAGUGACGAGCAAGUAUCCACUG

1598 HPV59_2757 CUUUCGCAGCGUUUAAGUGUGUUAC

1610 HPV59_1838 AUUAGUGAAGUUAUAGGGGAAACGC
1611 HPV59_1754 CCAGAUACGUGCAUGUUAAUUGAAC

1622 HPV59_1155 ACAGUAGUGAGAAAGCGGCGGCAGG
1623 HPV59_1130 CGAAAGUUUGGGUGCAGUAUAGAAA
1624 HPV59_1105 UGCACGGGAAAUGCAUGUUUUAAAA

1635 HPV59_459 AGGACAGUGUCGUGGGUGUCGGACC
1636 HPV59_379 CUAAAACCUCUAUGUCCAACAGAUA
1637 HPV59_354 GCUGCUGAUACGCUGUUAUAGAUGC

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 66 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs: 1647-1767 (See Table
13). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 66, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ

ID NOs: 1647-1767. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 66 comprising SEQ ID NOs:
1647-1767.

Table 13: Polyribonucleotide probes for determining HPV 66 nucleic acid.
SEQ ID
NO: Name Sequence 1658 HPV66_7377 GUGGUGUUCCUUACUGUUUAAUGUU

1683 HPV66_5999 GUCAUCCAUUAUUUAAUAGGCUGGA

1692 HPV66_5427 ACAGCUAAUGUUACUGCCCCUUUGG

1704 HPV66_4729 GUAGUACUACUAUAACAAACCCACU
1705 HPV66_4704 CCCACAUCUAGUACUGUACAUGUAA

1743 HPV66_1403 CACCAACACACCAAUUGCAGGAACU
1744 H PV66_1363 CACUCGGUAUCAAAUAUGGAUAUAG

1755 HPV66_759 UACCUUGUUGUAAGUGUGAGUUGGU
1756 HPV66_604 UAUAUUAGAACUUGCACCGCAAACG
1757 HPV66_579 GUAAAGUACCAACGUUGCAAGAGGU

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 68 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs: 1768-1875 (See Table
14). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV68, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 1768-1875. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 68 comprising SEQ ID NOs:
1768-1875.

Table 14: Polyribonucleotide probes for determining HPV 68 nucleic acid.
SEQ ID
NO: Name Sequence 1799 HPV68_6118 GGUACAUUACAAGAAACGAAAAGCG

1801 H PV68_6016 CCUACCAAUGUACAACAAGGGGACU

1812 HPV68_5245 UGGCUUCUGCUGCAUCCACUACAUA

1839 HPV68_3697 UGUUUCAGAAGCACAACGUGACAAG

1850 HPV68_2927 AGCCUUGCUAAAACUGCAUAUAGUG

1852 HPV68_2523 GUAUUUACAUAGUAGACUAACCGUG

1863 HPV68_1091 CAGACAGUAUAGAAAGCAGUCCUUU
1864 HPV68_897 UAAACAAACAGGUGACACAGUCUCA

In one embodiment, the present invention provides an isolated polynucleotide for specific hybridization to HPV 82 consisting essentially of a sequence or a complement thereof selected from the group consisting of SEQ ID NOs: 1876-2026 (See Table
15). In some embodiments, the present invention provides a set of polynucleotides for specific hybridization to HPV 82, wherein the set comprises at least one polynucleotide consisting essentially of a sequence or a complement thereof selected from the group consisting of: SEQ
ID NOs: 1876-2026. In certain embodiments, the methods of the present invention utilize a set of polynucleotide probes for specific hybridization to HPV 82 comprising SEQ ID NOs:
1876-2026.

Table 15: Polyribonucleotide probes for determining HPV 82 nucleic acid.
SEQ ID
NO: Name Sequence 1883 HPV82_7525 GGCAUAACCCUUAAUUCUUUUGGCA
1884 HPV82_7494 CAACUUUUGAACCACACUACCUAUG

1896 HPV82_6755 GGAUUCUACAAUUUUAGAACAGUGG

1908 HPV82_6137 UGUGUCUACUGUCAUUGAGGAUGGC

1919 HPV82_5467 GACACACAACAUGCUAUUGUUAUAC

1945 HPV82_4259 UUAUUCCUAAGGUAAAGGGCACUAC

1959 HPV82_3487 GACUCCUCCACAGUCACCCCGCUGU

1970 HPV82_2937 AUCAAAACAAAAGGCCUGCCAAGCC

1972 HPV82_2887 GAAAGAAACAUGCAAACCCUUAACC

1983 HPV82_1914 AGCACGUUUGAACUAUCGCAAAUGG
1984 HPV82_1889 ACAACUACAGCACAGUUUUGAUGAU
1985 HPV82_1841 CAUUAGUAGCACAUAUGGCGAAACA

1996 H PV82_1392 GACCUGGAAACAAACGAAAAUGCUA
1997 HPV82_1320 GAUGGGCAAAAUGACGGGUCACAAC

2009 HPV82_807 AUUUCAGCAAAUGUUACUGGGCGAC
2010 HPV82_782 AAAGCAGUGGAGACAGCCUUCGCAU

2021 HPV82_265 GGGACAAUACGCCAUAUGCAGCAUG
2022 HPV82_234 GUAGCAUUUACAGAACUUAGGAUUG
2023 HPV82_209 GUUGUGUAGAGCAGAUGUGUAUAAU

Hybridization The methods of the present invention comprises contacting the one or more polynucleotide probes with the sample under a hybridization condition sufficient for the one or more polynucleotide probes to hybridize to the target nucleic acid in the sample to form double-stranded nucleic acid hybrids. Preferably, the one or more polynucleotide probes is diluted in a probe diluent that also can act as a neutralizing hybridization buffer. The diluent can be used to dissolve and dilute the probe and also help restore the sample to about a neutral pH, e.g., about pH 6 to about pH 9, to provide a more favorable environment for hybridization. Sufficient volume of probe diluent, preferably one-half volume, can be used to neutralize one and one-half volume of base-treated sample. Preferably, the probe diluent is a 2-[bis(2-Hydroxyethyl) amino] ethane sulfonic acid (BES, Sigma, St. Louis, Mo.)/sodium acetate buffer. Most preferably, the probe diluent is a mixture of 2 M BES, 1 M sodium acetate, 0.05% of the antimicrobial agent NaN3, 5 mM of the metal chelating agent EDTA, 0.4% of the detergent TweenTM-20 and 20% of the hybridization accelerator dextran sulfate.
The pH of the probe diluent can be about 5 to about 5.5.
Thus, for example, after treatment with base, an aliquot of sample can be removed from the sample tube and combined with a sufficient amount of probe to allow hybridization to occur under a hybridization condition. The hybridization condition is sufficient to allow the one or more polynucleotide probes to anneal to a corresponding complementary nucleic acid sequence, if present, in the sample to form double-stranded nucleic acid hybrids. The probes and sample nucleic acids can be incubated for a hybridization time, preferably at least about 5 minutes, to allow the one or more polynucleotide probes to anneal to a corresponding complementary nucleic acid sequence. The hybridization condition can comprise a hybridization temperature of at least about 20 C, preferably about 50 to about 80 C. In certain embodiments, the hybridization is performed at a temperature of less than 55 C. In other embodiments when synRNA probes are used and when the sample containing the target nucleic acid contains a large volume of collection medium (i.e. > 1 ml), the hybridization temperature is between 45 C and 55 C and preferably is about 50 C (see figures 20A and 20B). Lowering the hybridization temperature provides the ability to detect 20,000 copies of HPV target nucleic acid in an assay. For any given target to be determined and the one or more polynucleotides employed, one of ordinary skill in the art can readily determine the desired hybridization condition by routine experimentation.
The present invention also allows for hybridization of probes to targets in the presence of anti-hybrid antibody that is immunospecific to double-stranded nucleic acid hybrids (i.e. the anti-hybrid antibody can be added at the same time or before the probes are added to the sample containing the target nucleic acid). This allows for reduction in the time to perform an assay.

Anti-hybrid Antibodies The double-stranded nucleic acid hybrids formed in accordance with the present invention can be detected using an antibody that is immunospecific to double-stranded nucleic acid hybrids. The antibody is immunospecific to double-stranded hybrids, such as but not limited to RNA/DNA; DNA/DNA; RNA/RNA; and mimics thereof, where "mimics"
as defined herein, refers to molecules that behave similarly to RNA/DNA, DNA/DNA, or RNA/RNA hybrids. The anti-double-stranded nucleic acid hybrid antibody (i.e., "anti-hybrid" antibody) that is utilized will depend on the type of double-stranded nucleic acid hybrid formed. In one embodiment, the antibody is immunospecific to RNA/DNA
hybrids.
It will be understood by those skilled in the art that either polyclonal or monoclonal anti-hybrid antibodies can be used and/or immobilized on a solid support or phase in the present assay as described below. Monoclonal antibody prepared using standard techniques can be used in place of the polyclonal antibodies. Also included are immunofragments or derivatives of antibodies specific for double-stranded hybrids, where such fragments or derivatives contain binding regions of the antibody.

For example, a polyclonal RNA:DNA hybrid antibody derived from goats immunized with an RNA:DNA hybrid can be used. Hybrid-specific antibody can be purified from the goat serum by affinity purification against RNA:DNA hybrid immobilized on a solid support, for example as described in Kitawaga et al., Mol. Immunology, 19:413 (1982);
and U. S.
Patent No. 4,732, 847, each of which is incorporated herein by reference.
Other suitable methods of producing or isolating antibodies, including human or artificial antibodies, can be used, including, for example, methods which select recombinant antibody (e.g., single chain Fv or Fab, or other fragments thereof) from a library, or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);
Jakobovits et al., Nature, 362: 255 (1993); and U.S. Pat. Nos. 5,545, 806 and 5,545, 807).
In one embodiment, the target nucleic acid to be determined is DNA (e.g., HPV

genomic DNA) or RNA (e.g., mRNA, ribosomal RNA, nucleolar RNA, transfer RNA, viral RNA, heterogeneous nuclear RNA), wherein the one or more polynucleotide probes are polyribonucleotides or polydeoxyribonucleotides, respectively. According to this embodiment, the double-stranded nucleic acid hybrids (i.e. DN/RNA hybrids) formed can be detected using an antibody that is immunospecific to RNA:DNA hybrids.
In a preferred embodiment of the present invention, a polyclonal anti-RNA/DNA
hybrid antibody is derived from goats immunized with an RNA/DNA hybrid. Hybrid-specific antibody is purified from the goat serum by affinity purification against RNA/DNA hybrid immobilized on a solid support. Monoclonal antibody prepared using standard techniques can be used in place of the polyclonal antibodies.
While any vertebrate may be used for the preparation of anti-RNA/DNA hybrid monoclonal antibodies, goats or rabbits are preferred. Preferably, a goat or rabbit is immunized with a synthetic poly(A)-poly(dT) hybrid by injecting the hybrid into the animal in accordance with conventional injection procedures. Polyclonal antibodies may be collected and purified from the blood of the animal with antibodies specific for the species of the immunized animal in accordance with well-known antibody isolation techniques. For the production of monoclonal antibodies, the spleen can be removed from the animal after a sufficient amount of time, and splenocytes can be fused with the appropriate myeloma cells to produce hybridomas. Hybridomas can then be screened for the ability to secrete the anti-hybrid antibody. Selected hybridomas may then be used for injection into the peritoneal cavity of a second animal for production of ascites fluid, which may be extracted and used as an enriched source of the desired monoclonal antibodies incorporated herein by reference.

In some embodiments, the step of detecting comprises contacting the double-stranded nucleic acid hybrids with a first anti-hybrid antibody to capture the double-stranded nucleic acid hybrids, wherein the first anti-hybrid antibody is immunospecific to double-stranded nucleic acid hybrids. In one embodiment, the first anti-hybrid antibody is immobilized onto a solid support such as a test tube surface. It will be understood by those skilled in the art that a solid support includes polystyrene, polyethylene, polypropylene, polycarbonate or any solid plastic material in the shape of test tubes, beads, microparticles, dip-sticks or the like.
Examples of a solid support also includes, without limitation, glass beads, silica beads, glass test tubes, and any other appropriate shape made of glass. A functionalized solid support such as plastic, silica, or glass that has been modified so that the surface contains carboxyl, amino, hydrazide or aldehyde groups can also be used. Immobilization of the antibody can be direct or indirect. Preferably, test tubes are directly coated with anti-hybrid antibody in accordance with methods known to those skilled in the art or briefly described below. The antibody can also be biotinylated and subsequently immobilized on, for example streptavidin coated tubes or silica, or modified by other methods to covalently bind to the solid phase.
Solubilized biotinylated antibody can be immobilized on the streptavidin coated tubes before capture of the hybridized samples as described below or in conjunction with the addition of the hybridized samples to simultaneously immobilize the biotinylated antibody and capture the hybrids.
In another embodiment, the first anti-hybrid antibody is attached to the solid phase in accordance with the method of Fleminger et al., Appl. Biochem. Biotech. 23:123 (1990), by oxidizing the carbohydrate portion of the antibody with periodate to yield reactive aldehyde groups. The aldehyde groups are then reacted with a hydrazide-modified solid phase such as MicroBind-HZTM microtiter plates available from Dynatech Laboratories (Chantilly, Va.).
Passive coating of the antibody according to the well known method of Esser, P., Nunc Bulletin No. 6 (November 1988) (Nunc, Roskilde, Denmark) can also be employed.
In other embodiments, Ventrex StarTM tubes (Ventrex Laboratories Inc., Portland, ME) are coated with streptavidin by the method of Haun et al., Anal. Biochem.
191:337-342 (1990). After binding of streptavidin, a biotinylated goat polyclonal antibody as described above, or otherwise produced by methods known to those skilled in the art, is bound to the immobilized streptavidin. Following antibody binding, tubes can be post-coated with a detergent such as TweenTM-20 and sucrose to block unbound sites on the tube and stabilize the bound proteins as described by Esser, Nunc Bulletin No. 8, pp. 1-5 (December 1990) and Nunc Bulletin No. 9, pp. 1-4 (June 1991) (Nunc, Roskilde, Denmark) and Ansari, et al., J.

Immunol. Methods, 84:117 (1985). Preferably, each tube is coated with between 10 ng and 100 g biotinylated antibody. Most preferably each tube is coated with approximately 250 ng of biotinylated antibody.
As discussed above, the solid phase can be coated with functional antibody fragments or derivatized functional fragments of the anti-hybrid antibody.
In some embodiments, hybridized samples are incubated in tubes coated with the first anti-hybrid antibody for a sufficient amount of time to allow capture of the double-stranded nucleic acid hybrids by the immobilized capture antibodies. The hybrids can be bound to the immobilized antibodies by incubation, for example incubation for about 5 minutes to about 24 hours at about 15 to about 65 C. In some embodiments, the incubation time is about 30 to about 120 minutes at about 20 to about 40 C, with shaking at about 300 to about 1200 rpm. In another embodiment, capture occurs with incubation at about one hour at about room temperature with vigorous shaking on a rotary platform. It will be understood by those skilled in the art that the incubation time, temperature, and/or shaking can be varied to achieve alternative capture kinetics as desired.
In other embodiments, the first anti-hybrid antibody is coupled to a magnetic bead (e.g., COOH-beads) to capture double-stranded nucleic acid hybrids. Magnetic bead-based technology is well known in the art. In some embodiments, magnetic silica beads having derivatized surfaces for reacting with antibody can be employed.
In one embodiment, the step of detecting further comprises providing a second anti-hybrid antibody that is immunospecific to double-stranded nucleic acid hybrids, wherein the second anti-hybrid antibody is detectably labeled either directly or indirectly.
For example, in some embodiments, an anti-hybrid antibody as described above can be conjugated to a detectable label to provide the second anti-hybrid antibody for detection of the double-stranded nucleic acid hybrids. Conjugation methods for labeling are well known in the art. Preferably, an antibody, such as the mouse monoclonal antibody deposited with the American Type Culture Collection as ATCC Accession number HB-8730, is conjugated to a detectable label such as alkaline phosphatase. It will be understood by those skilled in the art that any detectable label such as an enzyme, a fluorescent molecule, or a biotin-avidin conjugate can be used.
The antibody conjugate can be produced by well known methods such as direct reduction of the monoclonal antibody with dithiothreitol (DTT) to yield monovalent antibody fragments. The reduced antibody can then be directly conjugated to maleimated alkaline phosphatase by the methods of Ishikawa et al., J. Immunoassay 4:209-237 (1983) and Means et al., Chem. 1: 2-12 (1990), and the resulting conjugate can be purified by HPLC.
In another embodiment, the double-stranded nucleic acid hybrids can be detected indirectly, for example using an unlabelled anti-hybrid antibody for which a labeled antibody is specific. For example, the second anti-hybrid antibody can be a mouse immunoglobulin that is detected by a labeled goat anti-mouse antibody.
The double-stranded nucleic acid hybrids can be contacted with the second anti-hybrid antibody under a binding condition that is sufficient to provide for specific antibody-antigen binding (i.e., antibody/double-stranded nucleic acid hybrid binding), while minimizing non-specific binding. The binding condition preferably comprises a binding buffer comprising 0.1 M Tris-HC1, pH 7.5, 0.6 M NaCl to reduce cross reaction of antibody with other nucleic acid species, ZnC12 and MgC12 for stabilizing alkaline phosphatase, normal goat serum to block non-specific interaction of conjugate with the capture surface, 0.25% of the detergent TweenTM-20 to block non-specific binding of conjugate, and sodium azide as a preservative. Reactions can then be washed with a wash buffer (e.g., 0.1 M
Tris-HC1, pH 7.5, 0.6 M NaCl, 0.25% TweenTM-20, and sodium azide) to remove as much of the unbound or non-specifically bound second anti-hybrid antibody as possible. The second anti-hybrid antibody that is bound to the double-stranded nucleic acid hybrids can subsequently be detected, for example by colorimetry or chemiluminescence methods as described by e.g., Coutlee, et al., J. Clin. Microbiol. 27:1002-1007 (1989). For example, bound alkaline phosphatase conjugate can be detected by chemiluminescence with a reagent such as a Lumi-PhosTM 530 reagent (Lumigen, Detroit, MI) using a detector such as an E/LuminaTM
luminometer (Source Scientific Systems, Inc., Garden Grove, CA), an Optocomp ITM
Luminometer (MGM Instruments, Hamden, CT), or the like.
In some embodiments, the one or more polynucleotides can be conjugated to a label, such as an enzyme, or to a hapten such as biotin, that is then detected with a labeled anti-hapten antibody.
Thus, target-specific oligoribonucleotides or oligodeoxynucleotides can be designed using commercially available bioinformatics software. For example, for the detection of dsDNA targets, DNA can be denatured, hybridized to the RNA probes, and captured via anti-RNA:DNA hybrid antibodies on a solid support. Detection can be performed by various methods, including anti-RNA:DNA hybrid antibodies conjugated with alkaline phosphatase for chemiluminescent detection. Alternatively, other detection methods can be employed, for example using anti-RNA:DNA hybrid antibodies conjugated with phycoerythrin, suitable for detection by fluorescence.
In other embodiments, the methods of the present invention, optionally, further comprise a step of amplification of the target nucleic acid. Amplification techniques are known in the art and may be utilized. For example, Whole Genome Amplification (WGA) may be employed. WGA is an isothermal process that uses non-specific primers to generate amplicons using the target nucleic acid sequence as a template. For example, Phi 29 DNA
polymerase can be used in combination with non-specific primers to amplify target nucleic acid sequences. The polymerase can move along the target nucleic acid sequence displacing the complementary strand. The displaced strand becomes a template for replication allowing high yields of high-molecular weight DNA to be generated. For example, helicase-dependent amplification may be employed.

Kits In other aspects, the present invention provides a kit comprising the necessary components and reagents for performing the methods of the present invention.
The kit can comprise at least one of the following: an inert sample collection device, such as a dacron swab for exfoliated cell sample collection; a sample transport medium for stabilization of the sample during transport to the laboratory for analysis; a base, or a hydrolysis reagent; one or more polynucleotide probes specific for the target nucleic acid to be determined; neutralizing probe diluent; anti-hybrid antibody coated test tubes; and any necessary controls.
Preferably, the sample transport medium is Specimen Transport Medium ; the base is 0.415 M NaOH; the neutralizing probe diluent is a BES/sodium acetate buffer;
the test tubes are Ventrex StarTM tubes coated with a polyclonal anti-hybrid antibody; and the conjugated anti-hybrid antibody is a mouse monoclonal antibody conjugated to alkaline phosphatase.
Preferably, the kit also contains a substrate for the chemiluminescent detection of alkaline phosphatase, such as a CDP-Star with Emerald II (Applied Biosystems, Bedford, MA).
The present invention will be illustrated in more detail by way of Examples, but it is to be noted that the invention is not limited to the Examples.

EXAMPLES
Example 1: Polynucleotide probes for determining HPV 18 or HPV 16 DNA
Oligoarray 2.0 was chosen as the tool with which to identify RNA probes specific for HPV 18 or HPV 16 DNA. A database of sequences to be checked against, in this case, HPV

high risk and low risk types: 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89 was provided and the sequence of interest, i.e., HPV16 or HPV 18 was then BLASTed against the database to search for any regions of identity, and the similarities were stored.
Tm and %GC were then computed for ribonucleotides of a specified length and compared to the parameters, after which secondary structure was examined. Cross hybridization was checked with the Mfold package, using the similarity determined by BLAST.
The parameters of the Oligoarray 2.0 program were set to look for ribonucleotides of 25nt length, Tm range of 55-95 C, a GC range of 35-65%, and no secondary structure or cross-hybridization at 55 C or below. Using these parameters to determine ribonucleotide probes for HPV 18 (with a modified BLAST database that did not include HPV45, as we are not interested in specificity against that type) resulted in 145 ribonucleotides (for HPV 18) and 127 ribonucleotides (for HPV 16) covering a total of about 3.7kb of the target (i.e., HPV
18 or HPV 16 viral DNA). The sequences of the ribonucleotide probes that were selected are shown Tables 1 and 2 above. Sequence conservation across 20 HPV genomes is shown in Fig. Ia. As schematically shown in Fig. lb for HPV 18, all regions of the HPV
18 genome were represented in the respective probes.
RNA oligos were ordered from IDT technologies, at the 250 nM scale, with standard desalting. Oligos were stored in Ambion's RNA Storage Solution (1 mM Sodium Citrate, pH
6.4). The synthetic ribonucleotide probes are hereinafter referred to as "synRNA."

Example 2: Protocol for detecting HPV 18 DNA using HPV 18 synRNA
The hybridization and detection protocol was performed essentially as described in Table 16.
Table 16: Protocol Denature 1 Sample nucleic acid was denatured with alkali and heat.

2 Synthetic RNA probes were added to sample, hybridized, and neutralized 3 Synthetic RNA probe/target DNA hybrids were captured with Hybridize/ anti-hybrid antibody immobilized on a substrate Capture Conjugate 4 Alkaline phosphatase-conjugated anti-hybrid antibodies were added Samples were washed Wash 6 Alkaline-phosphatase-activated chemiluminescent substrate was Detection added Read 7 Samples were read using a luminometer Example 3: Results To remove as much variability as possible, data was analyzed as (S-N)/N, expressed as (S/N)-1. When signal = noise, data value = 0Ø

A. Specificity Demonstrated with HPV 18 synRNA
As shown in Table 17, the synthetic RNA probes (synRNAs) designed for HPV 18 showed no cross-reactivity with either HPV 6 or HPV 16 at up to 109 copies/assay (200 ng/ml). synRNA = 3.7kb coverage of HPV 18 DNA; 25 mers @ 1.34 nM final in hybridization.

Table 17: Specificity of HPV18 synRNA
Input copies Avg RLU S-N (SIN)-1 0 55 0 0.0 5000 167 113 2.1 = 10^4 238 183 3.4 10^5 2044 1989 36.5 0 53 0 0.0 co 10^7 79 26 0.5 2 10^8 59 6 0.1 10^9 84 32 0.6 0 51 0 0.0 10^7 51 0 0.0 a 10^8 54 3 0.1 10^9 60 9 0.2 B. Cross-reactivity of HPV 18 synRNA with HPV45 HPV 18 synRNA was not designed to be specific against HPV45 because HPV45 was not part of the specificity design. Accordingly, as shown in Table 18, synRNA
for HPV 18 showed cross-reactivity against HPV 45 plasmid only starting at between 106 and 107 copies of plasmid. synRNA = 3.7kb coverage of HPV 18 DNA; 25 mers @ 1.34 nM final in hybridization.
Table 18: Limited Cross-reactivity of HPV18 synRNA with HPV45 Input Copies Avg RLU S-N (S/N)-1 Oc 44 0 0.0 CO Z
5; O~ 2500 c 105 61 1.4 a .0 5000 c 111 67 1.5 1014 c 184 140 3.2 Oc 39 0 0.0 1015 c 51 12 0.3 1016 c 70 31 0.8 r' 1017 c 334 296 7.7 C. Determining Specificity with HPV 16 synRNA
As shown in Table 19, HPV16 synRNA is unable to detect HPVs 6, 18, or 45 at up to 109 copies/assay (200 ng/ml). synRNA = 3.175kb coverage of HPV 16 DNA; 25 mers 1.34 nM final in hybridization.

Table 19: Specificity of HPV16 s nRNA
Input Copies Avg RLU (S/N)-1 %CV
0c 24 0.0 5%
ao 5000 c 85 2.5 3%
= 10^4 c 157 5.5 3%
10^5 c 1270 51.4 2%
0c 24 0.0 0%
10^7 c 25 0.0 7%
= 10^8 c 24 0.0 2%
10^9 c 25 0.0 5%
0c 25 0.0 6%
U, 10^7 c 26 0.0 5%
= 10^8 c 28 0.1 17%
10^9 c 38 0.5 3%
0 c 29 0.0 33%
to 1OA7 c 24 -0.2 2%
= 10^8 c 26 -0.1 2%
10^9 c 24 -0.2 5%
D. Deterring different HPV Types About 0.5kb coverage of specific 25mer probes was provided for HPVs 16, 18, 31, and 45. As shown in Fig. 2, each HPV type was detected at 106 copies. synRNA
probes should be equally applicable to detection of whichever HPV types are desired.

E. Effect of synRNA coverage on sensitivity of detection Total coverage of synRNA probe affected signal in the assay. Increasing coverage improved signal in a non-linear fashion, probably due to base-stacking effects and loosening of secondary structure on the single-stranded DNA target as more synRNA probes are hybridized. As shown in Figure 3, at 3.7 kb of coverage, the sensitivity of detection was at 5,000 copies/assay.

F. Effect of synRNA concentration on sensitivity of detection As shown in Figure 4, increasing the concentration of synRNA increased sensitivity of detection. 25mer synRNA oligos had Tms about 45 to about 60 C. Increasing probe concentration raised that Tm, resulting in more efficient hybridization.
synRNA = 3.7kb coverage; 25mers @ concentrations shown in Figure 4.
G. Effect of synRNA size on sensitivity of detection As shown in Figure 5, given equivalent coverage, longer synRNA provided increased sensitivity.

H. Effect of synRNA contiguity on sensitivity of detection As shown in Figure 6, sensitivity increased as synRNA probes targeted adjacent regions. Without being held to a particular theory, it is believed that hybridization efficiency improved as the binding of one probe relaxed secondary structure on the target strand, providing a more accessible template for hybridization of the adjacent synRNA.
1. HPV 16 and HPV 18 are detected at equivalent levels As shown in Figure 7, HPV 16 synRNA, with about 3.175kb coverage, and HPV 18, with about 3.7kb coverage, gave about similar results. Both synRNAs were able to detect their respective targets at a concentration of 5,000 copies.

J. Comparison of different synRNA synthesis chemistries SynRNAs were prepared by TOM amidite chemistry (Operon Biotechnologies, Inc., Huntsville, AL) or by tBDMS chemistry (Integrated DNA Technologies (IDT)). As shown in Figure 8, 25mers of comparable quality can be provided using different chemical synthesis methods.

K. Detection at different temperatures With no RNA-dependant background occurring from synRNA, the hybridization temperature can be reduced, if desired, to provide a more tolerable condition for antibody/antigen interactions (Figure 9).

L. Exogenous RNase is unnecessary for detection synRNAs are largely devoid of secondary structure. This eliminates non-specific RNA-based background arising from anti-RNA:DNA hybrid antibodies recognizing long RNA secondary structures. With RNA not bound to DNA no longer contributing to background signal, the use of RNase A in the assay becomes unnecessary (Figure 10).

M. Discussion The method provided specificity and decreased background, and does not require RNase and is compatible with various media including SurePath, PC, STM and DCM.
The method provided a LOD with a 0.5kb target coverage is of 5pg/mL for HPV 18 with an S/N=3, whereas 2.5 kb target coverage could allow target detection to lpg/mL.
Example 4: Target capture and amplification The inclusion of a target amplification component provided enhanced sensitivity. The method detected as low as 10 copies of HPV plasmids or 10 SiHa cells comprising HPV
nucleic acid target. The method also provided robust specificity, the ability to distinguish HPV 16 or HPV 18 plasmid from all other high- and low-risk HPV types.
Target amplification can involve e.g., generating short amplicons with sequence-specific primers (e.g. Polymerase Chain Reaction) or large amplicons with multiple random primers (e.g. Whole Genome Amplification). Amplified targets can be captured and detected on a variety of different detection platforms.
Hybrid-specific antibodies were coupled to magnetic beads and employed in combination with short type-specific RNA probes for target capture. The sample processing procedure involved capture of targets pre-target amplification and the detection procedure involves capture of targets post-target amplification. To enhance assay sensitivity the isothermal WGA technology was utilized to produce non-specific amplification of any captured targets.
The nucleic acid target of interest was immobilized on a solid support with the use of type-specific RNA probes to form nucleic acid hybrids and anti-RNA:DNA hybrid-specific antibodies to capture, concentrate and purify. The sample preparation process produced single-stranded DNA targets free of amplification inhibitors and non-specific targets and allowed for multiple targets to be captured simultaneously. This was demonstrated by coupling hybrid capture antibodies to magnetic beads and using HPV sequence-specific RNA
probes for detection.

Magnetic beads coupled with anti-hybrid antibodies were used to specifically capture amplicons generated by WGA. Short RNA probes were used for specific detection.
In addition, anti-RNA:DNA hybrid antibodies coupled with alkaline phosphatase was used for detection.
Table 20 shows a flowchart representing a method steps in accordance with one embodiment. Detection reagent 1 is preferably the detection reagent 1 provided in the digene Hybrid Capture Kit and detection reagent 2 is preferably the detection reagent 2 provided in the digene Hybrid Capture Kit. Detection Reagent 1 comprises alkaline phosphatase-conjugated antibodies to RNA:DNA hybrids and Detection Reagent 2 comprises CDP-Star with Emerald II (chemiluminescent substrate).
Table 20. Protocol.
Assay Flow Chart Target Denaturation RNA probe hybridization and capture with anti-hybrid antibody Wash Isothermal Amplification Amplicon Denaturation RNA probe hybridization and capture with anti-hybrid antibody Detection Reagent 1 Wash Detection Reagent 2 One hundred (100) copies of HPV 18 plasmid are obtained after 30 minutes of WGA
(Fig. 11) Five hundred (500) copies of HPV 18 plasmid are detected after 15 minutes of WGA;
and detection of 1000 copies of HPV18 plasmid are obtained after only 10 minutes of WGA
(Fig. 12).
Ten (10) copies of plasmid or 10 SiHa cells comprising HPV nucleic acid are detected with longer amplification times of 45 minutes or greater (Fig. 13).
Figure 14 shows specificity for HPV 18.

The results demonstrated that after 45 minutes of amplification, as little as 10 copies of plasmid or 10 SiHa cells can be detected; and about 1000 copies of plasmid can be detected after only 10 minutes of amplification.

Example 5: Synthetic type-specific biotinylated DNA probes DNA probes.
In another embodiment, synthetic type-specific biotinylated DNA probes are used to form double-stranded hybrids with target mRNA (Fig. 15). Hybrids are captured on magnetic streptavidin beads. Signal amplification and detection is performed with anti-hybrid antibody/alkaline phosphatase and the resulting chemiluminescent signal is detected.
Example 6: Sample Assay Flow.
Predenatured samples are transferred to a multiwell plate. Probes in neutralizing solution are added to the denatured sample and incubated with shaking at room temperature for about 1 minute to neutralize the sample. The neutralized samples are transferred to a plate containing immobilized anti-RNA:DNA hybrid antibodies so that target DNA
is allowed to hybridize to the synthetic RNA probes and also to be captured by the immobilized antibodies. The incubation is at about 55 C for about 120 min. Anti-RNA:DNA
hybrid antibodies conjugated with alkaline phosphatase are added at room temp and incubated for about 30 min. After the conjugated antibody step, the plate is washed for about 12 min. A
dioxetane substrate is added and incubated for 15 minutes. The plate is then read with a luminometer.
Hybridization and hybrid capture by anti-RNA:DNA hybrid antibodies are performed in the same step at about 55 C and may include shaking.

Claims (17)

1. A method for determining the presence of a target nucleic acid in a sample, the method comprising:
a) contacting one or more polynucleotide probes with the sample under a hybridization condition sufficient for the one or more polynucleotide probes to hybridize to the target nucleic acid in the sample to form double-stranded nucleic acid hybrids, wherein the one or more polynucleotide probes does not hybridize to a variant of the target nucleic acid; and b) detecting the double-stranded nucleic acid hybrids, wherein detecting comprises contacting the double-stranded nucleic acid hybrids with a first anti-hybrid antibody that is immunospecific to double-stranded nucleic acid hybrids, whereby detection of the double-stranded nucleic acid hybrids determines the target nucleic acid in the sample.
2. The method of Claim 1 wherein the detecting further comprises providing a second anti-hybrid antibody that is immunospecific to double-stranded nucleic acid hybrids, wherein the second anti-hybrid antibody is detectably labeled.
3. The method of Claim 1 wherein the at least one probe and the anti-hybrid antibody are added in the same step.
4. The method of Claim 1, wherein the target nucleic acid is an HPV nucleic acid.
5. The method of Claim 4, wherein the HPV nucleic acid is HPV DNA of a high risk HPV
type.
6. The method of Claim 5, wherein the HPV type is HPV 16, wherein the variant is a nucleic acid of a type selected from the group consisting of: HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
7. The method of Claim 5, wherein the HPV type is HPV 18, wherein the variant is nucleic acid of a type selected from the group consisting of: HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
8. The method of Claim 5, wherein the HPV type is HPV 45, wherein the variant is nucleic acid of a type selected from the group consisting of. HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 16, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, and 89.
9. The method of Claim 5 wherein the HPV type is a hrHPV type and wherein the variant is a nucleic acid of low risk HPV type.
10. The method of Claim 5, wherein the one or more polynucleotide probes consist essentially of a sequence or a complement thereof selected from the group consisting of SEQ
ID NOs: 1-2026.
11. A method for determining the presence of HPV 18 DNA in a sample, the method comprising:
a) contacting one or more polynucleotide probes or a complement thereof with the sample under a hybridization condition sufficient to allow the one or polynucleotides to anneal to a corresponding complementary nucleic acid sequence in the sample to form double-stranded nucleic acid hybrids, wherein the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs:163-309; and b) detecting double-stranded nucleic acid hybrids, whereby detection of the double-stranded nucleic acid hybrids indicates the presence of HPV 18 DNA in the sample.
12. A method for determining the presence of HPV 16 DNA in a sample, the method comprising:
a) contacting one or more polynucleotide probes or a complement thereof with the sample under a hybridization condition sufficient to allow the one or polynucleotides to anneal to a corresponding complementary nucleic acid sequence in the sample to form double-stranded nucleic acid hybrids, wherein the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs:1-162; and b) detecting double-stranded nucleic acid hybrids, whereby detection of the double-stranded nucleic acid hybrids indicates the presence of HPV 16 DNA in the sample.
13. A method for determining the presence of HPV 45 DNA in a sample, the method comprising:
a) contacting one or more polynucleotide probes or a complement thereof with the sample under a hybridization condition sufficient to allow the one or polynucleotides to anneal to a corresponding complementary nucleic acid sequence in the sample to form double-stranded nucleic acid hybrids, wherein the one or more polynucleotide probes is a set of nucleic acid probes comprising at least one nucleic acid sequence chosen from the group consisting of. SEQ ID NOs:842-974; and b) detecting double-stranded nucleic acid hybrids, whereby detection of the double-stranded nucleic acid hybrids indicates the presence of HPV 45 DNA in the sample.
14. A probe set selected from the group consisting of SEQ ID NO; 1-162 (HPV
16); 163-309(HPV 18); 842-974(HPV 45); 310-454(HPV 31); 455-579(HPV 33); 580-722(HPV
35);
723-841(HPV 39); 975-1120(HPV 51); 1121-1252(HPV 52); 1253-1367(HPV 56); 1368-1497(HPV 58); 1498-1646(HPV 59); 1647-1767(HPV 66); 1768-1875(HPV 68); and 2026(HPV 82).
15. The method of claim 1 wherein the one or more polynucleotide probes is a mixture of probe sets comprising the probes set forth in SEQ ID NO: 1-2026.
16. The method of claim 1 wherein the hybridization is performed at about 45 to about 55 °C.
17. A kit comprising the probe set of Claim 14.
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