AU2010356288B2 - Methods and kits for detecting an infectious agent - Google Patents

Abstract

The present invention provides methods and kits for determining the presence, absence, or level of an infectious agent in a sample. Specifically, the present invention provides methods and kits for detecting or quantifying certain target polynucleotides of the infectious agent. In certain embodiments, the present invention provides for such detection without the need for amplification (e.g., replication) of the target molecule and/or without the need for labor intensive purification procedures. In certain embodiments, the present invention provides positive control and housekeeping gene for normalization and quantatively detection of the copy numbers of infectious agent in a sample. In these or other embodiments, the invention allows for such detection with the desired sensitivity and/or specificity, even where the polynucleotide is present in the sample at low copy number.

Description

PCT/US2010/040649 WO 2012/002964

METHODS AND KITS FOR DETECTING AN INFECTIOUS AGENT

FIELD OF THE INVENTION

[0001I This invention relates to detecting the presence, absence, or level of an infectious agent in a sample.

BACKGROUND OF THE INVENTION

[0002] Although many tests have been developed to diagnose infection, many of these tests are only specific and/or sensitive when there is a high load of the infectious agent present, i.e. in the ease of late or advanced stages of infection. In addition, some of these tests require highly technical personnel or specialized knowledge to carry out the tests.

[0003] For example, to date, there is no robust test available for detecting high risk strains of HPV in pap smears, and which is both clinically sensitive and specific, as well as convenient, easy and inexpensive, without the need for highly trained personnel. (Kurtycz et al., Comparison of Methods Trial for High-Risk HPV Diagnostic Cytopathology 38:1 ΟΤΙ 08 (2009); Ginocchio et al., Comparison of the Third Wave Invader Human Papilloma (HPV) Assay and the Digene HP V Hybrid Capture 2 Assay for Detection of High-Risk HPV DNA J Clin. Microbiol. 46:1641-1646 (2008)). The Pap or Papanicolau test is currently the test of choice for the initial screening of pre-malignant or malignant cervical cancers caused by HPV. The Pap test is a cytology test which requires highly trained personnel to discriminate between normal cells and cells undergoing malignant lesions under a light microscope. The Diagene-HC2 DNA test which is the only FDA-market approved HPV test, relies on the capture of DNA/RNA hybrids on a solid phase. The captured DNA/RNA hybrids are detected using an enzyme-linked antibody upon amplification. Although the test can detect up to thirteen HPV types, it has poor sensitivity (5000 copies/mL) and specificity. Furthermore, the test requires purification of DNA from the sample and it cross-hybridizes with low-risk and high-risk HPV types when the viral load in the sample is high.

[0004] Other tests for HPV include GenProbe’s Aptima HPV test, Third Wave’s Invader HPV DNA test, Ventana's Inform! HPV in-situ hybridization test, and other PC’R-based tests, including Roche’s Amplicor or Linear Array HPV DNA tests. These tests require purification of the nucleic acid from the samples and/or amplification of the target nucleic PCT/US2010/040649 WO 2012/002964 adds prior to detection, which can result in poor sensitivity or specificity, in addition, these tests can be expensive and time-consuming. Furthermore, since most of these tests require amplification of the target polynucleotides, a highly clean and dedicated environment is required to ensure that there is no carry o ver or cross contamination between samples.

[0005] Accordingly, there is a need for an efficient, rapid, reliable, and inexpensive method and/or assay for detecting an infectious agent, such as HPV. and which has the desired level of sensitivity and specificity for screening and/or evaluating samples for the presence or level of the infectious agent.

SUMMARY OF THE INVENTION

[0006] The present invention provides methods and kits for determining the presence, absence, or level of an infectious agent in a sample. Specifically, the present invention provides methods and kits for detecting or quantifying certain target polynucleotides of the infectious agent, in certain embodiments, the present invention provides for such detection without the need for amplification (e.g., replication) of the target molecule and/or without the need for labor and/or time intensive procedures. In these or other embodiments, the invention allows for such detection with the desired sensitivity and/or specificity, even where the polynucleotide is present in the sample at low copy number.

[0007] In one aspect, the invention provides a method for detecting the presence, absence, or level of an infectious agent in a sample, such as a biological sample. The method comprises capturing a target polynucleotide, if present, from the sample, where the target polynucleotide is indicative of the presence of the infectious agent. The target polynucleotide is then detected directly or indirectly by hybridization with one or a series of signal-amplifying polynucleotide probes (e.g., branched DNA). The method allows detection of the target polynucleotide, with the desired specificity and/or sensitivity, and at low copy number, to thereby allow for detection of even early stage infection.

[0008] The target polynucleotide may be captured from the sample by hybridization to a Capture Extender probe having at least a first sequence and a second sequence, the first sequence hybridizing to the target polynucleotide and the second sequence hybridizing to an immobilized capture probe. The capture probe may be immobilized via any solid support (e.g., chip, bead, or well). ? PCT/US2010/040649 WO 2012/002964 [0009] The captured target polynucleotide is then detected by hybridizing one or a series of signal-amplifying polynucleotide probe(s) directly or indirectly to the target. The series of signal-amplifying probes may comprise branched DNA. For example, the series of signal-amplifying probes may comprise a pre-Amplifier probe, an Amplifier probe, and a Label probe. Specifically, the pre-Amplifier probe hybridizes to the target through a Label Extender probe. The Label Extender probe generally has a first sequence and a second sequence, the first sequence hybridizing to the target polynucleotide and the second sequence hybridizing to the pre-Amplifier probe. Consecuti ve hybridizations of Amplifier probes and Label probes may then be conducted to amplify the signal. The signal-amplifying probes may allow for amplification of the signal 40 times or more, and in some embodiments, 200 times or more (e.g., relati ve to the number of hybridized Label Extender probes).

[00101 in various embodiments, the target polynucleotide or biomarker is a polynucleotide involved in the replication machinery' of the infectious agent (e.g,, cis- or trans-acting factor), a polynucleotide encoding a protein involved in replication or transcription, or a polynucleotide encoding a cell surface protein, integral membrane protein, etc. For example, in the case of HPV the target polynucleotides may be independently selected from E6 and E7 sequences. Exemplary Capture Extender and Label Extender probes for various infectious agent targets, including HPV, HBV, HIV, influenza H1N1, HCV, SARS, Mycobacterium, Syphilis, Aspergillus, Candida, and Cryptococcus are described herein.

[0011] in another aspect, the present invention provides a kit for detecting target polynucleotides. The kit provides one or more Capture Extender probes and one or more Label Extender probes for the detection of particular target polynucleotide(s), and exemplary pairs or “sets” of Capture Extender probes and Label Extender probes are disclosed herein. In some embodiments, the kit may further comprise one or more capture probes, a solid support, and/or a signal amplifying probe set (e.g., the set comprising a pre-Amplifier probe, an Amplifier probe, and a Label probe). The kit may comprise (in addition to the Capture Extender and Label Extender probes for detecting the target polynucleotide), Capture Extender and Label Extender probes for detecting one or more positive and/or negative control sequences. The positive control probes may be used to normalize between samples and/or to calculate the copy number of polynucleotides present. Exemplary positive control probes may comprise sequences from housekeeping genes, such as but not limited to a I veer aldehyde 3-phosphate dehydrogenase (GAPDH/GAPD).

[001 ΙΑ] In another aspect, the invention also provides a method for determining the presence, absence, or level of HPV type-16 in a sample, consisting of the following steps: 2010356288 07 Apr 2017 a. capturing a target polynucleotide, if present, from a sample by hybridisation to a set of capture extender oligonucleotides probes consisting of SEQ ID NOs: 14 to 21 and wherein the target polynucleotide is indicative of the presence of said HPV type 16; b. hybridising said target polynucleotide with a set of blocking oligonucleotides probes consisting of SEQ ID NOs: 38 to 48; and c. detecting the target polynucleotide by hybridisation with a set of label extender oligonucleotides probes consisting of SEQ ID NOs: 22 to 37; and wherein said probes in steps (a), (b) and (c) for detecting HPV have a sensitivity of about 3000 copies.

[0012] Other aspects and embodiments of the invention will be apparent to the skilled artisan in view of the following detailed description.

[0012A] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0012B] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 (A, B, and C) illustrates the components of the test system for detecting target polynucleotides in connection with the invention.

[0014] FIG. 2 shows a standard curve for detection of HPV type 16 ssDNA. 4 [0015] FIG. 3 shows the relative Iijght units (itLU) emitted front Alkaline Phosphatase-labeled probe, ibr detecting HPV biomarkers in Pup smears of HPV patients during various disease slates. AUCL'S is atypical squamous cells of undetermined significance. CIN is cervical intraepithelial neoplasia. 13 K. is background. 2010356288 07 Apr 2017 [tKtlti] FIG. 4 shows detection of HI V target polynucleotides that are present in amounts as low as 25 copies. | m> L 71 FIG. 5 shows a standard curve for the detection for HBV polynucleotides.

[QQ18] FIG. fi shows a standard curve for the detection of HCV (top panel), and quantitation of HCV particles from sera of six patients (bottom panel). FIG. 7 shows a standard curve for the detection of SARS. FIG. 8A and B shows standard curves for the detection of ssDNA complementary to 18S RNA from fungal cells {Candida albican*). |IHI2I| FIG. 9 shows quantifying different species and strains of Aspergillus and Candida. j(Kt22J FIG. 10 shows quantifying Candida from three clinical blood samples.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides methods and kits for determining the presence, absence, or level of an infectious agent in a sample. Specific ally, the present invention provides methods and kits for detecting or quantifying certain target polynucleotides of the infectious agent. In certain embodiments, the present invention provides for such detection SW ll-t NO BLOCKING t.ABLi. fcXi LNDbk NOCLtOllDH SEQUENCt Ob GAPDH a?i. ;:> q,i: m ^aaiat cc aqtflab r-afj £7-¾ capc(Stct!Ct(i^gtjjg^g t5ctc<?]ft(!aa;iiea£iec 100871 According to another aspect, the present invention provides a kit for detecting target polynucleotides, for example, associated with a pathogenic organism. The kit comprises at least one Capture Extender probe and at least one Label Extender probe specific for a target polynucleotide from an infectious agent as described above. The kit may comprise a set of

4A PCT/US2010/040649 WO 2012/002964 without the need for amplification (e.g., replication) of the target molecule and/or without the need for labor and/or time intensive procedures, such as nucleic acid purification steps. In these or other embodiments, the invention allows for rapid detection of the target, with the desired sensitivity and/or specificity, even where the polynucleotide is present in the sample at low copy number.

[0024] In one aspect, the invention provides a method for detecting the presence, absence, or level of an infectious agent, such as a biological sample. The method comprises capturing one or more target polynucleotide/s), if present, from the sample, wherein the target polynucleotide is indicative of the presence of the infectious agent. In one embodiment, a single polynucleotide of interest is detected (single plex detection). In another embodiment, two polynucleotides of interest are detected (two-plex detection), or more than two polynucleotides of interest are detected (multiplex detection). The target polynucleotide is then detected directly or indirectly by hybridization with one or a series of signal-amplifying polynucleotide probes. The method may further comprise detecting the level of one or more control polynucleotides, for example, to normalize signals across samples.

[0025j For example, the target polynucleotide may be captured by hybridization to a Capture Extender probe having at least a first sequence and a second sequence, the first sequence hybridizing to the target polynucleotide and the second sequence hybridizing to an immobilized capture probe. The capture probe may be immobilized via any solid support, including but not limited to a chip (e.g., an array), well, bead, or other solid support or matrix.

[0026] The captured target polynucleotide is then detected by hybridizing one or a series of signal-amplifying polynucleotide probes, either directly to the target polynucleotide, or indirectly through a Label Extender probe. A Label Extender probe generally has a first sequence and a second sequence, the first sequence hybridizing to the target polynucleotide and the second sequence hybridizing to a signal-amplifying polynucleotide probe. The signal-amplifying probe may comprise branched DMA, e.g., may include a pre-Amplifier probe, an Amplifier probe, and a Label probe.

[0027] The sample may be a liquid sample, or a biological sample, such as, for example, a body fluid. In various embodiments, the sample is blood, plasma, serum, urine, vaginal secretion, pap smear, semen, nasal swabbing, lung lavage, pleural effusion, sputum, and throat swab. In other embodiments liquid samples can be obtained from swabs of objects aiii'ii min seats, pond water, tree sap, and insect or plant extracts. Other types of samples 5 PCT/US2010/040649 WO 2012/002964 for which detection of infectious agents is desired may be employed in connection with the invention. In certain embodiments, a nucleic acid purification step may be performed prior to detection, such as the use of any commercially available filter-based method or kit for purification of nucleic acids, in other embodiments, no such purification step is required.

[0028} The method of detection may he carried out using the principles set forth in U.S. Patent Number 7,709,198, which is hereby incorporated by reference. For example, detection generally takes place with the use of a series of signal amplifying polynucleotide probes (e.g., branched DNA), which is hybridized directly to the target polynucleotide, or indirectly through a series of hybridization reactions. The signal-amplifying probes obviate the need for thermal cycling or amplification of the target sequences prior to detection. Thus, in such embodiments, the method intensifies the signal of hybridization by multiple layers of probe hybridization, instead of any actual nucleotide sequence amplification of the target polynucleotide.

[0029] In various embodiments, the target polynucleotide (which may be linear or circular) is captured on a solid support by hybridizing to one or more sets of probes. The set of probes generally comprises a Capture Extender and a Label Extender, and optionally a Blocking Label Extender (or blocking probe).

[0030] The Capture Extender indirectly captures the target polynucleotide by hybridizing, simultaneously, to the target polynucleotide and a Capture Probe attached to a solid support, e.g., bead, chip, etc. The Capture Extender generally has a first sequence that hybridizes to the target polynucleotide and a second sequence that hybridizes to the Capture Probe. The Capture Extender optionally comprises a linking sequence between the first sequence and the second sequence. The first sequence of the Capture Extender, which hybridizes to the target polynucleotide, is generally from about 15 to about 30 nucleotides in length, or about 20 to about 27 nucleotides in length. The sequence and length is generally selected to hybridize under the hybridization conditions selected or described herein. The second sequence of the Capture Extender, which may hybridize to the capture probe, may be any capturable sequence, but is generally selected to hybridize under the selected hybridization conditions. The capture sequence is generally about 10 to about 30 nucleotides in length, and exemplary' capture sequences are disclosed herein. The linking sequence is selected to provide for the physical independence of the first and second sequences, and is selected to a void significant 6 PCT/U S2010/040649 WO 2012/002964 structural constraints. The linking sequence may be short, for example, from about 2 to about 10 nucleotides. The linking sequence may be poly(T) orpoIy(A) in certain embodiments. j 00311 Various Capture Extender sequences (and sets of Label Extender probes) are disclosed herein for the detection of numerous infectious agents. See Tables 3, 6, 9, 12, 15, 18, 21,24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, and 93. It is understood that various modifications of these sequences may be made within the spirit of the invention, such as the addition or deletion of from 1 to 4 nucleotides to any or each of the target-hybridizing sequence, capture sequence, and/or linking sequence, or the addition of from 1 to 5 degenerate nucleotides.

[0032] A Blocking Label Extender may optionally be employed to block certain sequences. Such optional blocking probes (and sets of Blocking probes) for use with the invention are described in Tables 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, and 95. it is understood that various modifications of these sequences may be made within the spirit of the invention, such as the addition or deletion of from 1 to 4 nucleotides, or the addition of from 1 to 5 degenerate nucleotides.

[0033] The Label Extender hybridizes to the target polynucleotide and a signal-amplifying polynucleotide simultaneously. For example, the Label Extender generally comprises a first sequence that hybridizes to the target sequence, and a second sequence that hybridizes to a signal amplifying probe. The Label Extender may optionally have a linking sequence. The first sequence which hybridizes to the target sequence may generally be from about 15 to about 30 nucleotides in length, or about 20 to about 27 nucleotides in length. The sequence and length of the first sequence is generally selected to hybridize under the hybridization conditions selected or described herein. The second sequence of the Label Extender, which may hybridize to the signal amplifying probe, is generally selected to hybridize under the selected hybridization conditions. The second sequence is generally about 10 to about 30 nucleotides in length, and exemplary sequences are disclosed herein. The linking sequence is selected to provide for the physical independence of the first and second sequences, and is selected to avoid significant structural constraints. The linking sequence may be short, for example, from about 2 to about 10 nucleotides. The linking sequence may be poiy(T) or poly(A) in certain embodiments. PCT/US2010/040649 WO 2012/002964 [0034] Various Label Extender sequences (including Label Extender probe sets) for the detection of certain infectious agents are disclosed herein. See Tables I, 2, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, and 94. It is understood that various modifications of these sequences may he made within the spirit of the invention, such as the addi tion or deletion of from 1 to 4 nucleotides to any or each of the target-hybridizing sequence, capture sequence, and/or linking sequence, or the addition of from 1 to 5 degenerate nucleotides. 10035] The hybridization conditions for the assay include selected temperature, time, and reagents so as to allow for rapid, sensitive, and/or specific detection. For example, the hybridization temperatures may be independently selected in the range of about 45°C to about 65°C, or about 50°C to about 60°C, such as about 55°C, The hybridization buffer is generally 3x 5SC. or comparable hybridization buffer.

[0036] The length of time for hybridization of the Capture Extender and the Label Extender probes may be selected based on the type of virus suspected, the stage of infection, the copy number of virus present in the sample, etc. In some embodiments, the Capture Extender and Label Extender probes are allowed to hybridize to the target for at least about 5 minutes, such as within the range of about 10 minutes to about 120 minutes, such as about 20 to about 90 minutes, or about 30 or 60 minutes. In certain embodiments, the Capture Extender and Label Extender probes are allowed to hybridize to the target for about 60 minutes to about 4 hours, 8 hours, 12 hours or overnight (e.g,, from 18 to 24 hours). With samples suspected of representing early stages of an infection, (or where the copy number of target polynucleotides is expected to be low), the Capture Extender and the Label Extender probes are allow to hybridize to the target for a longer period of time, than for samples suspected of representing advanced stages of infection (or where a high copy number of the target polynucleotides is expected). For example, the hybridization time may be from about 1 hour to about 4 hours, from about 3 hours to 8 hours, from about 7 hours to about 12 hours, or from about 12 hours to about 24 hours or longer for samples obtained from the early stages of infection or where the copy number of infectious agen ts are low. Where the copy number of infectious agents are high or during the late or advance stages of infection, the length of time needed for the probes to hybridize to the target polynucleotide may be from about 5 minutes to about one hour. PCT/US2010/040649 WO 2012/002964 [0037] A robust detection of the hybridization reactions is obtained by either directly detecting the multiple hybridizations of Label Extenders to the target polynucleotide, or by further hybridization of one or a series of signal-amplifying probes. For example, detection may occur by hybridizing a pre-Amplifler probe to the label extender probe, and hybridizing Amplifier probes to the pre-Amplifier probe, which complex may be detected with Label probes. Such reactions may take place in consecutive hybridizations, e.g., at conditions in the range of 40 to 60° C, in 3x SSC (or comparable hybridization buffer), and for about 30 to about 60 minutes each. Exact conditions may be selected based upon the sequences and melting temperature of each probe. Exemplary sequences/structures for the pre-Amplifier probe, Amplifier probe, and label probes are known. For illustration, an exemplary sequence of the preamplifier is 5' AGGC AT AGGAC CCGTGT CTtttttttt ttAGG C ATAGG ACC CGT GTCTtttttAT GCTTT G ACT CAG AAAACGGTAACTTC 3’ (SEQ ID NO:l). The underlined sequences are complementary to sequences in the GAPD Label Extenders (used as a positive control as described herein).

[0038] The configuration and hybridization of test components, such as Label Extenders, may be as described in U.S. Patent No 7,709,198, which is hereby incorporated by reference. For example, Label Extenders may be described with reference to the “cruciform” configuration or the “double Z” configuration. See FIG. 1C, Generally, one end of the Label Extender hybridizes to the preamplifier probe, and the other end hybridizes to the target polynucleotide. The pre-amplifier probe may hybridize to a single Label Extender probe as described herein (e.g., cruciform configuration). In some embodiments using the double Z-configuration, the preamplifier probe hybridizes to two Label Extender probes.

[0039] An exemplary Label Extender probe set in the cruciform configuration is set-forth in Table 1 and Table 94, for glyceraldehyde 3-phosphate dehydrogenase (GAPD), which maybe used as a control for normalizing across samples, and for quantifying polynucleotide copy-number.

[0040] The sequences complementary to the preamplifier sequence of SEQ ID NO: 1 is un derlined for each of the Label Extenders shown in Table 1.

Table 1. GADP Label Extenders with Cruciform Configuration

SEQ ID NO: PROBE LABEL EXTENDER NUCLEOTIDE SEQ IDENTIFIER OF GLYCERALDEHYDE 3-PHOSPHATE 9 PCT/U S2010/040649 DEHYDROGENASE SEQ ID NO:2 GAPD 127 ccagtggactccacgacgtacTTTTTeaasttaccetttt CPI tail SEQ ID NO :3 GAPD 128 crgaatcaaascatTTTTTttctccatgstgfitgaagacg CP2 head SEQ ID NO:4 GAPD 129 tcttgaggctgitgtcatacttctTTTTTgaasitaccgttti CPI tail SEQ ID NO :5 GAPD 130 ctgagtcaaagcatTTTTTgcaggaggcattgctgatga CP2 head SEQ ID NO:6 GAPD 131 cagtagaggcagggatgatgftcTTTTT gaagttaccgtttt CPI tail SEQ ID NO :7 GAPD 132 cteaetcaaaecatTTTTTcacagccttggcaecgc CP2 head WO 2012/002964 [0041] Table 2 shows an exemplary Label Extender probe set for GAPD in the double Z configuration. The sequences complementary to the preamplifier sequence is underlined for each of the Label Extenders shown in Table 2.

Table 2. GAPD Label Extenders with Double Z configuration SEQ ID NO: PROBE IDENTIFIER. LABEL EXTENDER NUCLEOTIDE SEQ OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE SEQ ID NO :8 GAPD217 ccagtggactccacgacgiacTTTTTgaagttaccgtttt CPI tail SEQ ID NO :9 GAPD218 ttctccatggtggtgaagacgTTTTTctsastca aascat CP2 tail SEQ ID NO: 10 GAPD219 tcitgaggctgttgtcatacttcfTTTTTgaagttaccgtttt CPI tail SEQ ID NO: 11 G.APD220 gcaggaggcattgctgatgaTTTTTctgagieaaagcai CP2 tail SEQ ID NO: 12 GAPD221 eagtagaggcagggatgatgtteTTTTTgaagtiaccgtttt CPI tail SEQ ID NO: 13 GAPD222 cacagccttggeagcgcTTTTTctgagtcaaagcat CP2 tail [0042] The Amplifier probe or Label probe, in particular embodiments, is conjugated with Alkaline Phosphatase, allowing its detection with glow luminescence. Alternatively, an Amplifier probe or label probe may have a chromogenic or fluorescent generating moiety (e.g., enzyme) to provide an intensified signal in the presence of the appropriate substrate. An exemplary format is shown in Figure 1, illustrating an Alkaline Phosphatase Label probe and corresponding luminescent substrate.

[0043] The number of Amplifier probes that hybridize to the growing complex, per the number of hybridized pre-Amplifier probes, may be within the range of 5:1 to 20:1. The number of Label probes that hybridize to the growing complex, per the number of hybridized Amplifier probes, may be within the range of 2:1 to about 5:1.

[0044] The Capture Extender and the Label Extender are designed to comprise at least one sequence that, recognizes (hybridizes) to the target polynucleotide. The level of 10 PCT/US2010/040649 WO 2012/002964 complementarity is appropriately selected based upon the desired hybridization conditions and selectivity, and in some embodiments, the complementary sequence is fully complementary to its intended target. In certain embodiments, the Capture Extender and/or Labe! Extender includes from 1 to about 5 degenerate nucleotides in the target-hybridizing sequence, so as to provide for the desired selectivity or cross-reactivity.

[0045] The Capture Extender and Label Extender may be designed to recognize and bind to such target sequences as inverted terminal repeats (e.g., of a virus genome); bacterial 5S, 16S, or 23S ribosomal RNA sequences, terminal inverted repeat sequences (TIR), miniature inverted repeat transposable elements (MITE), CpG sequences in prokaryotic cells such as bacteria; or 18S or 28S ribosomal RNA sequences in eukaryotic cells, such as fungi or parasites.

[0046] In various embodiments, the sensitivity of the detection is less than about 1000 copies, less than about 500 copies, less than about 250 copies, or less than about 100 copies (e.g., about 25 copies) of the target sequence. The full test may take less than about 7 hours, and in some embodiments, may take less than about 6 hours, or less than about 5 hours.

[0047] Detection moeities for the target polynucleotide and controls may be the same or different, and may be independently selected from a luminescence-generating moiety, such as but not limited to alkaline phosphatase having a luminescent substrate, and a chromogenic generating moiety, such as but not limited to a horseradish peroxidase (HRP). In some embodiments, detection of the one or more target polynucleotides may be carried out simultaneously or sequentially with the detection of the positive control or normalization control (e.g., GAPDH), that is, in the same or parallel reaction. In some embodiments, detection signals for the target polynucleotide and the normalization control may be read sequentially from a patient sample in the same reaction. In such embodiments, after detection of a luminescent or chromogenic reaction, the sample is washed, and a different luminescent or chromogenic reagent is added.

[0048] In various embodiments, the target polynucleotide is indicative of the presence of a vims of the family Paramyxoviridae (e.g,, parainfluenza, mumps, measles); Orthomyxoviridae (e.g., influenza); Hepadnaviridae (e.g·., hepatitis); Adenoviridae (e.g., acute respiratory disease); Poxviridae (e.g,, small pox); Herpesviridae (e.g., HSV, Varicella Zoster, Karposi sarcoma); Papillomaviridae (e.g., HPV); Polyomaviridae (e.g., cystitis or mi'M r»r nmite respiratory diseases); Parvoviridae; Rhabdoviridae (e.g., rabies); Filoviridae 11 PCT/U S2010/040649 WO 2012/002964 (e.g., hemorrhagic fever caused by Ebola virus and Marburg virus); Bunyaviridae (e.g., encephalitis, Hantavirus respiratory syndrome, Rift Valley fever); Arenaviridae (e.g., aseptic meningitis, encephalitis, meningoencephalitis, Lassa fever); Coronaviridae (e.g., severe acute respiratory syndrome or SARS); Flaviviradae (e.g., Dengue hemorrhagic fever); Togaviridae; Picomaviridae; Caliciviridae (e.g., winter vomiting disease); Astroviridae (e.g., gastroenteritis); Retroviridae (e.g., HIV, HTLV) and Reoviridae (e.g., Colorado Tick fever). 10049 [ in certain embodiments, the target polynucleotide is an HPV target polynucleotide, and may be a high risk type HPV, such as type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, or 68; or may be a low risk type HPV, such as type 6, 11, 40, 42, 43, or 44. High risk sexually transmitted HPV may lead to development of cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia (AIN), whereas low risk sexually transmitted HPV may lead to genital warts and are not life-threatening since they do not lead to cervical cancer. Of the high risk type HPV, types 16 and 18 are the most dangerous since they cause about 70% of cervical cancer. For example, in a NIH study, it was found that 10% of women infected with type 16 or 18 HPV developed advance precancerous cervical disease (CIN3) within 3 years while 20% did so in 10 years, [0050] The invention in certain embodiments allows for the determination of a specific type of HPV present in a sample, and/or distinguishes between early course of the infection, and late or latent stage of HPV infection.

[0051] In various embodiments, the target polynucleotide sequence is a portion of an HPV late viral gene (e.g., LI and/or L2), or an early viral gene (e.g., El and/or E2), which encode proteins responsible for replicating and maintaining the viral DNA as a circular episome. In one exemplary embodiment, the target polynucleotide is a portion of the gene encoding the E6 and/or E7 proteins of the HPV. In particular, the target polynucleotides are derived from the mRNA regions encoding the E6 and/or E7 proteins that mediate degradation of the tumor suppressors, p53 and retinoblastoma (RB), by interfering with the regulation of the cell cycle.

[0052] In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence of HPV type 16. In such embodiments, at least one Capture Extender (e.g., at least two, three, four or fU/irt comprise a target-hybridizing sequence selected from Table 3 (underlined). At 12 PCT/US2010/040649 WO 2012/002964 least one Capture Extender may comprise, consist essentially of, or consist of a sequence independently selected from SEQ ID NOS: 14 to 21 (Tables 3). In these or other embodiments, at least one Label Extender (e.g., at least two, three, four, or five) comprises a target-hybridizing sequence selected from Table 4 (underlined). At least one Label Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 22 to 37 (Table 4). The method may further employ a Blocking Label Extender {BE), e.g., at least one, two, or three BLs each comprising, consisting essentially of, or consisting of a sequence selected from SEQ ID NOS: 38 to 48 (Table 5),

Table 3: Capture Extender of HPV type 16 SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF HPV TYPE-16 14. ctcctuteuatcctaaaacattTTTTTctcttggaaagaaagt 15. cacgtcgcagtaactattacttTTTTTctcttggaaagaaagt 16. tgttgUccatacaaaciataacaataaiTTTTTctcltggaaagaaagt 17. aatciaacatatattcatgcaatataggTTTTTcicttggaaagaaagt 18. atattetaataeectctetcceTTTTT ctcttggaaagaaagt 19. cccattaacaggtcticcaaaetaTTTTTctcttggaaagaaagt 20. netaeattatcetttctcasaacaeatTTTTTctcttggaaagaaagt 21. SEQ ID NO cccgtaccctcttccccattTTTTTctcttggaaggaaagt Table 4: Label Extender of HPV ty pe 16 LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HPV TYPE-16 22. ate tacttttatactaacc ggtttcTTTTT gaagttaccgtttt 23. scaEttetcttttsEtseataaaatTTTTTctgagtcaaagcat 24. aactgtagtaactttctssEtegTTTTTgaagttaccgtttt 25. 26. asttEttt sea Ectct EtscatTTTTT ctgagtcaaagcat ttcccatctctatatactatgcataaatTTTTTgaagttaccgtttt 27. aacatttatcacatacagcatatggaTTTTTctgagtcaaagcat 28. 29. cggtttgttgtattgctgttctaaTTTTTgaagttaccgtttt taatacacctaattaacaaateacacaaTTTTTctgagtcaaageat 30. ggacacagtggettttgacagtTTTTT gaagttaccgittt 31. ccaeatetctttecttttcttcaTTTTTctgagtcaaagcat 32. aatctecaacaasacatacatceaTTTTT gaagttaccgittt 33. tgggtttctctacgtgttcttgatTTTTT ctgagtcaaagcat 34. gagatcagttgtctctggttgcaTTTTT gaagttaccgtttt 35. gctgtcatttaattgctcataacagtaTTTTTctgagtcaaagcat 36. tcacacttacaacaaaaggttacaTTTTT gaagttaccgtttt 37. cgcacaaccgaagcgtagagTTTTTctgagtcaaagcat 13 PCT/US2010/040649

Table 5: Blocking Label Extender of HPV type 16 SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HPV TYPE-16 38. gcagtacacacattctaatattatatcatgtat 39. c-ccgaaaagcaaagtcatatacct 40. gtctatactcactaattttagaataaaacttta 41. ttatattatggaatctttgctttttgt 42. ccggtccaccgacccc 43. tgtatciccatgeatgattacagc 44. catctatttcatcctcctcctctga 45. gttctgcttgtccagctggac 46. cgaatgtctacgtgtgtgc tttgta 47. ggggcacacaattcctagtgtg 48. ggtacctgeaggatcagceat WO 2012/002964 [00531 In certain embodiments, the target polynucleotide is an E6 or E7 sequence from HPV type 18. In such embodiments, at least one Capture Extender probe (e.g, at least two, three, four, or five) comprises a target-hybridizing sequence selected from Table 6 (underlined). Thus, at least one Capture Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 49 to 56 (Tables 6). At least one Label Extender probe (e.g,, at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 7 (underlined). At least one Label Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 57 to 72 (Table 7). The test may further employ Blocking Label (BL), e.g., at least one, two. or three BLs each containing a sequence selected from SEQ ID NOS: 73 to 75 (Table 8).

Table 6: Capture Extender of HPV type 18 SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF HPV TYPE-18 49. cetttttcattaaeetetctaagtttitTTTTTctettggaaagaaagt 50. tgctcggttgcagcacsTTTTTctcttggaaagaaagt 51. tgttgccttaggtccatgcataTTTTTctettggaaagaaagi 52. gctttctactactascttaattciescTTTTTctcttggaaagaaagt 53. ctggatcagceattgttgcttTTTTTctcttggaaagaaagt 54. tactacctactaatcttctatgagcttTTTTTctcttggaaagaaagt 55. tggccagggaagatceactTTTTTctcttggaaagaaagt 56. ttecaectcaatacaaaacgeTTTTTctcttggaaagaaagt 14 PCT/US2010/040649

Table 7: Label Extender of HPV ty pe 18 SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HPV TYPE-18 57. ccca gctatgttgtggaatcgt'TTTTT gaagttaccgtttt 58. aatggeactggeetctatagtgTTTTTctgagtcaaagcat 59. tcgttggagtctttcctgtcgTTTTTgaagttaccgtttt 60. cttaatattatacttgtgtttctctgcgTTTTTctgagtcaaagcat 61. taaatgcaatacaaigicttgcaaTTTTTgaagltaceglttt 62. ggaatttcattttggggctcTTTTTctgagtcaaageat 63. gctcgtgacatagaaggtcaaccTTTTTgaagttaccgtttt 64. tttcttcctctgagtcgcttaattTTTTTctgagtcaaagcat 65. atgattaactccatctatttcatcgtlTTTTgaagttaccgtttt 66. 67. 68. cgtcgggctggtaaatgttgTTTTTctgagtcaaagcat sctcaaaggtcgtcigctgaTTTTTgaagttaccgittt tgttcagaaacagctgctggaatTTTTT ctgagtcaaagcat 69. cggacacaeaaaggacagggTTTTTgaagtiaecgtttt 70. actactgggatgcacaccaTTTTTctgagtcaaagcat 71. cgtcagtttcctcatctgaaaacitTTTTTgaagttaccgtttt 72. cgtcaagteatttaagcttggtaccTTTTTctgagtcaaagcat Table 8: Blocking Label Extender of HPV ty pe 18 SEQ ID NO 73. BLOCKING LABEL EXTENDER NUCLEOT IDE] SEQUENCE OF HPV TYPE-18 tgtgtgac gttgtggtteagct 74. ttcacacttacaacacatacacaacat 75. gaattaggagtcagcagtcattattc WO 2012/002964 [0054] in certain embodiments, the target polynucleotide is an E6 or E7 sequence from HPV type 31. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target-hybridizing sequence selected from Table 9 (underlined). Thus, at least one Capture Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 76 to 82 (Tables 9). At least one Label Extender probe (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 10 (underlined). At least one Label Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 83 to 94 (Table 10). The test may' further employ Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 95 to 99 (Table 11), PCT/US2010/040649

Table 9: Capture Extender of HPV type 31 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 31 76. &amp;cttictgcaggatttttgaacTTTTTctcttggaaasaaagt 77 gcttagttcatgcaatttccgagTTTTTctcttgsaaagaaagt 78. cctctgtttctattaactgacctttgTnTTctcttggaaagaaagt 79. atctaaattcacttacttttgaataaaatTTTTT ctcttggaaagaaagt 80. catatacctttgtttgtcaatttttctTTTTT ctettggaaagaaagt 81. acgcatgtttacacttgggtttTTTTTctcttggaaagaaagt 82. tctgagctgtcgggiaattgcTTTTT ctcttggaaagaaagt Table 10: Label Extender of HPV type 31 SEQ ID NO LABEL EXTENDER OF HPV TYPE 31 83. gtagggtatttccaatgccgaTTTTTgaagttaccgtttt 84. cagtagacacaattcaatcttagttcatcTTTTTctgagtcaaagcat 85. gtggtgtgtcgtccctatatactattTTTTTgaagttaccgtttt 86. 87. cttaaacattttgtacacactccgtTTTTTctgagtcaaagcat acacgttatacacctaattaacaaatcaTTTTTgaagttaccgtttt 88. tcttctggacacaacggtctttgTTTTTctgagtcaaagcat 89. tctttttatccaaatgtctttgttttTTTTTgaagttaccgtttt 90. 91. ttcctcctatgttgtggaatcgttTTTTTcigagtcaaagcat cttgcaacgtaggtgtttctecTTTTTgaagltaccgttt-t 92. ctcaggttgcaaatctaacacatagtTTTTTctgagtcaaagcat 93. ggactgtctatgacatcc-tcctcaTTTTTgaagttaccgtitt 94. cca sttctecttstccagctinTT ctgaeicaaagcat Table 11: Blocking Label Extender of HPV type 31 SEQ ID NO BLOCKING EXTENDER OF HPV TYPE 31 95. gttaaatetglaaatgcaaaatctaata 96. Q7 aatgitgrtccatacacactatatctatacc ctatgcaacglcctgtocacc 98. cagtacgaggicttctccaacatg 99. tcataacagtggaggtcagttgc WO 2012/002964 [0055] In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from HPV type 35. In such embodiments, at least one Capture Extender probe {e.g., at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 12 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 100 to 105 (Tables 12), In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 13 (underlined). At least one Label Extender nrnbe may comprise, consist essentially of, or consist of a sequence selected from 16 PCT/US2010/040649 WO 2012/002964 SEQ ID NOS: 106 to 117 (Table 13). The test may further employ a Blocking Label (BE), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 118 to 127 (Table 14).

Table 12: Capture Extender of HPV type 35 SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF HPV TYPE-35 100. eaaacaaatttcatggatsctttcTTTTTctcttggaaagaaagt 101. tactccatateecteeccttcTTTTTctcttggaaagaaagt 102. tetttttctaacatttctccatacacTTTTTctcttggaaagaaagt 103. caccgtccaccgatgttatgTTTTTctcttggaaagaaagt 104. tccagctggaccgtcaatagtatnTTTctcttggaaagaaagt 105. SEQ ID NO ccat taataaatcttccaat ttacgtaTTTTT ctcttggaaa gaaagt Table 13: Label Extender of HPV type 35 LABEL. EXTENDER NUCLEOTIDE SEQUENCE OF HPV TYPE-35 106. gcagtttgtaaggtcgttcagctTTTTTgaagttaccgtttt 107. ttctacctcgttgcacaaatcatTTTTTctgagtcaaagcat 108. taattcttgtttgcagtatacacaatt'TTTTTgaagttaccgtttt 109. aaagtcatatacctcactccgctgTTTTTctgagtcaaagcat 110. 111. aataaatgacataactgtttgttgcatTTTTTgaagttaccgtttt ggtttttgacatgtaatacacctaattTTTTTctgagtcaaagcat 112. tctaaaacatagtrttgcaatgtagttatTTTTTgaagttaccgtttt 113. agttgcctcgggttccaaaTTTTTctaagtcaaaacat 114. acacaattgctcataacaetatageicTTTTTgaagtiaccettti 115. 116. cttc ctcctcc t cteascts! cTTTTT ctgaetcaaagcat ggaggtgtctggttttgcttgTTTTT gaagttaccstttt 117. ttacaacaggacgttacaatattataattTTTTTctgagtcaaagcat Table 14: Blocking Label Extender of HPV type 35 SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HPV TYPE-35 118. ictatatactatacacaaatcatagcatgc 119. gaataaaattttaaacatttcatgca 120. actaiatctataccatctatattcacttattttt 121. ttgcttttcaactggacacagc 122. gaatcgttttttttcttctaaatgtct 123. 124. aacaggacatacaccgaectgtc ggtttctctacgtgttggtttec 125. ttctccatgcatgattacacctc 126. cagacgtagtgtcgcctcacat 127. tgtcaatgtgtgtgctctgtacaca 17 PCT/US2010/040649 WO 2012/002964 [0056] in certain embodiments, the target polynucleotide is an E6 or E7 sequence from HPV type 39, In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target-hybridizing sequence selected from Table 15 (underlined). Thus, at least one Capture Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 128 to 134 (Tables 15). At least one Label Extender probe (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 7 (underlined). At least one Label Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 135 to 145 (Table 16). The test may further employ Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 146 to 154 (Table 17).

Table 15: Capture Extender of HPV type 39 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 39 128. tacetcggttteetetagtggTTTTT<rtcttggaaagaaagt 129. ggttccccgtccctatatactaTTTTTclettggaaagaaagt 130. ttiateaaatcticeiitsctatiiTTTTTcicttggaaagaaagt 131. aatccacccatatcteatsitataTTTTTctcitsgaaagaaagL 132. tttcctgcaaggtgggctttTTTTTctcttggaaaeaaagt 133, ccgtgaggcttctactaccagctTTTTTctcttggaaagaaagt 134. SEQ ID NO acaaatc«tagtgagtccataaacagTTTTT ctcttggaaagaaagt Table 16: Label Extender of HPV type 39 LABEL EXTENDER OF HPV TYPE 39 135. 136. cccgtattttagcataaaattttataTTTTT ctgagtcaaagcat 137. 138. ttagttatattttctaatgtagttgcatacaTTTTTctgagtcaaagcat cacagcggtttcagaeaacacaTTTTTgaagttaccgtttt 139. 140. aggtgtcttaatttttctsctggaTTTTTctgagtcaaagcat ttcattgtaageacataaaictaatacaaTTTTTgaagttaccgtttt 141. catacaaggtcaaccggctgtatTTTTT ctgagtcaaagcat 142. gtcgggttcatctatttcatcctTTTTTgaagttaccgtttt 143, ttgatgttggtgattaactgcatgTTTTTctgagtcaaagcat 144. ggttcateccgtctggctagtagTTTTTgaagttaccgtttt 145. gaacactgtattgtgtgaegCTgtTTTTTctgagtcaaagcat 18 PCT/US2010/040649

Table 17: Blocking Label Extender of HPV type 39 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 39 146. ^ 147. tgcaccttattaataaattatataactttgta 148. actgtc ctgtatagcttcct get at 149. tggtccagcaccgtcgac 150. tgcggtcctcccgttttg 151. cttgggtttctcttcgtgttagtc 152. ctgactcteetaattgctcgtga 153. geagtgtgttgttacacttaeaacac 154. ctgctgtagttgtcgcagagtatc WO 2012/002964 [0057] In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from HPV type 45. In such embodiments, at least one Capture Extender probe (eg., at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 18 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 155 to 160 (Tables 18). In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 19 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 161 to 172 (Table 19). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 173 to 180 (Table 20).

Table 18: Capture Extender of HPV type 45 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 45 155. tatacctctgtgcgttccaaiatTTTTTctcttggaaagaaagt 156. tataaataaatctttaaaascaaattgaTTTTTctcttggaaagaaagt 157. caatsttsctcegeatcca'TTTTTctcttggaaagaaagt 158. tgaciaacgccatctgcttcatTTTTTctcttggaaagaaagt 159. 160. agctcaattctsccgtcacacTTTTTctcttggaaagaaagt catctgcc ga get ctctact gtaTTTTT ctcttggaaagaaagt 19 PCT/US2010/040649

Table 19: Label Extender of HPV type 45 SEQ ID NO LABEL EXTENDER OF HPV TYPE 45 161. tttctgctgggttcaatggttTTTTTsraagttaccgtttt 162. cgttfgtcxttaaggtgtctacgttTTTTTctgagtcaaagcat 163. tgtattacactgccctcagtactsTTTTTgaagttaccgtttt 164. gccgtgcctggtcacaacaTTTTTctgagtcaaagcat 165. cctacstctgcsaastctttcttTTTTTgaagttaccetttt 166. tgcatacttottgctatecttgtgtttcTTTTT ctgagtcaaagcat 167. ttcc aaatgcaatacaaittetigTTITTgaagttaccgtitt 168. 169. caacaggatctaattcattctgaggTTTTTctgagtcaaagcat ttaattgctcgtaacacaacaggtTTTTTgaagttaccgtttt 170. cgttticctcctctgactcgcTTTTT ctgagtcaaagcat 171. tcgggctggtagttgigcaTTTTTgaagttaccgtttt 172. cgctgtggttcggctcgTTTTTctgagtcaaagcat Table 20: Blocking Label Extender of HPV type 45 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 45 173. geagcatatgctatacagtctctatacac 174. ggaataaaagtctatacatttatggcat 175. gagtttgaataatatcttaattctctaattct 176. ttttttccagtgtctctccataiaca 177. cttattaacaaattatacaactctgtattagtta 178. tc Iggcaccgcaggcac 179. tccagctatgctgtggaatctt 180. ttacaacatacacacaaaattttgtga WO 2012/002964 [0058] in certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from HPV type 51. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 21 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 181 to 187 (Tables 21). In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 22 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 188 to 201 (Table 22). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 202 to 206 (Table 23). 20 PCT/US2010/040649

Table 21: Capture Extender of HPV type 51 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 51 181. ggtctttccctctoitcttcaaaTTTTTctcttggaaagaaagt 182. tgcategaaacgticaaagcttcTTTTT ctcttggaaagaaagt 183. tttgaaiaaaacagtaaacattgtttgTTTTTctcttggaaagaaagt 184. tcgataaatcatataagctttttttagiaaTTTTTctcttggaaagaaagt 185. cgcattaccccetccaaTTTTTctettagaaagaaagt 186, cgtgtacgttgccagcaattagTTTTTctcttggaaagaaagt 187. SEQ ID NO ggagetteaattctgtaacacgtaTTTTT ctcttggaaagaaagt Table 22: Label Extender of HPV type 51 LABEL EXTENDER OF HPV TYPE 51 188. ttacaatacacacacactacctgtatattgTTTTT gaagttaccgtttt 189. ttalatacatctectetacataattcctttTTTTTctgagtcaaagcat 190. 191. atacaatcttaatttcagtaaatgctacaTTTTTgaagttaccgtttt catactgcatatggattettatccctatTTTTTctgagtcaaagcat 192. acctgctataacgtctatactctctaattTTTTTgaagttaccgtttt 193. ttgcctctaatgtagtaccatacacagTTTTTctgagtcaaagcat 194. gtggtctttgacatctatgacaccttaTTTTTgaagttaccgtttt 195. tttgcttttcttcaaacccaaTTTTTctgagtcaaagcat 196, cacttgggtttcgttacgttgtTTTTTgaagttaccgtttt 197. cattaccacgcatggctttattaTTTTTctgagtcaaagcat 198. tacaataciaeatetittaattgtsgtaTTTTTgaaRttacegtttt 199. agteaatitcagtctatggtattaaaTTTTTctgagteaaageat 200. teaaattgctcatagcattgcaTTTTTgaagttaecgtttt 201. tacttcatcctcctcctctgagctgTTTTTctgagtcaaagcat Table 23: Blocking Label Extender of HPV type 51 SEQ ID NO BLOCKING EXTENDER OF HPV TYPE 51 202. acataattcatgcagcgttcgt 203. ct ttttttttcgtc cac c a art 204. cgtcccgctatttcatggaac 205. ctggtagctggtcacgcatattatc· 206, gcctgtccagcccgtcttt. WO 2012/002964 [0059] In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from HPV type 52, In such embodiments, at least one Capture Extender probe (e.g, at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 24 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from 8EQ ID NOS: 207 to 212 (Tables 24). In such 21 PCT/US2010/040649 WO 2012/002964 embodiments, at least one Label Extender (e,g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in "fable 25 (underlined). At least one Label Extender probe may comprise, consist essentially oil or consist of a sequence selected from SEQ ID NOS: 213 to 224 (Table 25). The test may further employ a Blocking Labe! (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 225 to 238 (Table 26).

Table 24: Capture Extender of HPV type 52 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 52 207. stecaeeatccggegtcTTTTTctcttggaaagaaagt 208. cactetgaacagcgccctgtTTTTT ctcttggaaagaaagt 209. cacaggtcggggtctccaaTTTTTctcttggaaagaaagt 210. cagtgctatgaatgcatagccgTTTTT ctcttggaaagaaagt 211. tgtgcccaacagcatttgcTTTTTctcttggaaagaaagt 212. ttcagggtcctccattgcasTTTTTctcttggaaagaaagt

Table 25: Label Extender of HPV type 52 SEQ ID NO LABEL EXTENDER OF HPV ’TYPE 52 213. cticcagcacctcacacaattcTTTTTgaagttaccgtttt 214. gccttaittcatgcaecaattTTTTTctgagtcaaagcat 215. ttieeaetgcaeacactgcaTTTTTgaagttaccgtttt 216. taiacetcicncgitgtagctctttTTTTTcigagicaaagcat 217. tttctttttcttcaggacataatggTTTTTgaagttaccgtttt 218. teactigiitgcattaaeaigtcTTTTTctgagtcaaagcat 219. cgcatgacgttacacttgggtTTTTTgaagttaccgiUt 220. aatettttataatisctttgtctccaTTTTTctgagtcaaagcat 221. accgeiccacaecatctetatTTTTTgaagttaccgttti 222. scttgttctgettgtccatctgTTTTTctgagtcaaagcat 223. atatcacaatetagtaaUgcttgtgTTTTTgaaettaccgtttt 224. tagtgtgctatcacaactgtgaeaatTTTTTctgagteaaagcat

Table 26: Blocking Label Extender of HPV ty pe 52 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 52 225. eiattegtaaatcigtaaatagaaacttg 226. cgccatatggattattgtctctatata 227. aaaagcgtaggcacataatacaca 228. tgataatgcetatattcacttatettagata 229. tctaatgttttcecataeagtgaatat 230. cacttaatggtttttttaccctctct 231. cgtttgacaaattatacatctaatagttaltt 232. ccaacgacccataatatiatgaaa O'X'X ttgtttcaggttgcagatctaatatat 22 PCT/U S2010/040649 SEQ ID NO BLOCKING LABEL EXT ENDER OF HPV TYPE 52 234. aatigcicatagcagtgtaggicag 235. ccteclcatc tgagctgtcacci 236. tgtagagtacgaagglcegtcg 237. ccggggcacacaacttgtaa 238. ggttgtttatagccgtgcacag WO 2012/002964 [0060] In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from HPV type 56. In such embodiments, at least one Capture Extender probe (e.g, at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 27 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 239 to 245 (Tables 27). In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 28 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 246 to 257 (Table 28). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 258 to 262 (Table 29).

Table 27: Capture Extender of HPV type 56 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 56 239. gttagttcttttttgcaatatacacatgTTTTT ctcttggaaagaaagt 240. atgcaaaattatatacctcagcacgtTTTTT ctcttggaaagaaagt 241. gcacactgcataaggaaaatcaTTTTTctcttegaaagaaagt 242. cacaatgcaattgcttttcctcTTTTTctcttggaaagaaagt 243. ctattagateaaatcgtctttttctgtTTTTT ctcttggaaagaaagt 244. gtctccagcaccccaaacatTTTTTctcttggaaagaaagt 245. aggtcaatttctgtttgaegtgttaTTTTTctcttggaaagaaagt Table 28: Label Extender of HPV type 56 SEQ ID NO LABEL EXTENDER OF HPV TYPE 56 246. catacactgaatagtcataatacctatatttTTTTTgaagttaccgtttt 247. gttttttagttatactttctaatgtagctcTTTTTctgagtcaaagcat 248. gtagcaccttattaataaatcacataactTTTTTgaagttaccgtttt 249. cggagttaacggactttgacatcf ITTITctgagtcaaagcat 250. tgtagattctctaggttctetagatgttfnTTI’gaagttaccgtttt 251. ttggtacittaccaigcatsatialacTTTTTctgagtcaaagcat 23 PCT/US2010/040649 SEQ ID NO LABEL EXTENDER OF HPV 'ΓΥΡΕ 56 252. cctcatecteatcctetgaactTTTTTgaagttaccKtttt 253. sctcctaeaaatggtetacttcatTTTTTctgagtcaaagcat 254. cttgtctagcttgctgtggccTTTTTgaagttaccgtttt 255. gtgtattaegtaacacgtatcttctttaoTTTTTctgagtcaaagcat 256. cacaaacttacactcacaacaaggtacTTTTTgaagttaccgtttt 257. ggtactgtgaatgtccaactgcacTTTTTctaagtcaaagcat Table 29: Blocking Label Extender of HPV type 56 SEQ ID NO BLOCKING LABEL, EXTENDER. OF HPV TYPE 56 258. tccctatacactaagtttaattcagigc 259. tctaactttactataaaacaataaacatactct 260. gacccggtccaaccaigtg 261. gttctaatataacgtcitgeagcg 262. gtccaattgcicattgcaetgt WO 2012/002964 [0061| In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from HPV type 58. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 30 (underlined), At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 263 to 269 (Tables 30). In such embodiments, at least one Label Extender (e.g·., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 31 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 270 to 281 ('fable 31). The test may further employ a Blocking Label (BL), e.g,, at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 282 to 286 (Table 32).

Table 30: Capture Extender of HPV type 58 SEQ ID NO 263. CAPTURE EXTENDER OF HPV TYPE 58 ctcctctgcatcctaaaacaTTTTTctcttggaaagaaagt 264. 265. 266. tcaattcgatttcatacacagaTTTTTctcttggaaagaaagt gtctttttgcattcaacgcattTTTTTctettgeaaagaaaat cactattcttaaatctgcaaatacaaagTTTTTctcttggaaagaaagt 267. 268. agcaatcgtaaacacactttacatacTTTTT etcttggaaagaaagt attaatatttcatttaaacacttttttagtatTTTTT etcttggaaagaaagt 269. ccgtccaagcctatttcatcctTTTTTctcttgeaaagaaaet 24 PCT/US2010/040649

Table 31: Label Extender of HPV type 58 SEQ ID NO LABEL EXTENDER OF HPV TYPE 58 270. atcatacaatgtccstsatttTTTTT gaagttaccgtttt 271. tgtciccaacecctgacacaaTTTTTctgagtcaaagcat ! £, ataattataatatctatactcacttattttagatTTTTTeaacttaccgtttt 273. ttettetaatstetctccatataecsaTTTTTctgaetcaaagcat 274. caateatctttsacaaataatacatctaTITTTgaagttaccgtttt 275. tgcctttttttttcttgtgeaca 11111 ctgagtcaaagcat 276. cctgtceaacgacccgaaatat Γ1 T 1 T gaagttaccgtttt 277. tccaaeaeactgcacagcgcTTTTTctgagtcaaagcat 278. tirtstttatciacstcggsgtcTTTTT gaagttaccgtttt 279. atsecgttgltacaegttacactTTTTTctgagtcaaagcat 280. tcataecagaataggtcaettgatTTTTTgaagttaccgttlt 281. cgtctgagctgtcacataattgcTTTTTctgagtcaaagcat Table 32: Blocking Label Extender of HPV type 58 SEQ ID NO BLOCKING LABEL EXTEN DER OF HPV TYPE 58 282. t c atat ac ct c agate gctgcaa a 283. tgcaaatggatttccatctctata 284. tatgaaaccttttgtttaaatccaca 285. gcgttgggttgtttcctctc 286. tcaggatgtaaaictaaaatatattctctta WO 2012/002964 100621 In certain embodiments, the target polynucleotide is an 126 and/or E7 sequence from HPV {ype 59. In such embodiments, at least one Capture Extender probe (e.g. at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 33 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 287 to 292 (Tables 33). hi such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 34 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 293 to 304 (Table 34). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 305 to 317 (Table 35). PCT/US2010/040649

Table 33: SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 59 287. ggatcctcaaagcgtgcca'TTTTTctcttggaaagaaagt 288. tgatgcgaatatcatgcagaggTTTTTctcttggaaagaaagt 289. tctataacagcgtatcaecasctcTTTTT ctcttggaaaeaaagt 290. igtiggacaiagaggatiaggca IT i' l IVienggauagauagi 291. tagstetcttscteggetccTTTTT ctcttggaaagaaagt 292. aaagtgttgcttttggtecatgTTTTTctcttggaaagaaagt

Table 34: Label Extender of HPV type 59 SEQ ID NO LABEL EXTENDER OF HPV TYPE 59 293. cccctttgcaaaacacacaatTTTTTeaagttaccgtttt 294. tcaaatacctctetttcttgcagttTTTTTctgagtcaaagcat 295. cctctaatgtttctccatacacggTTTTTgaagttaccgtttt 296. atgtaacggtgtcttggttteagTTTTTctgagtcaaagcat 297. gcgcttgtcgttgctgtctTTTTTgaagttaecgtttt 298. cattgttttacaccagtgtttcactacTTTTTctgagtcaaagcat 299. ggttccaaatciaaaaeaatocacTTTTTgaagttaccgtttt 300. aaggtcaacttcctcataattttgtTTTTTctgagtcaaagcat 301. tcaggtaatigctcgtagcacacTTTTTgaagttaccgtttt 302. ttttcattctcggagtcggagTTTTTctgagtcaaagcat 303. gcgaggtttctactactagctgaagTTTTTgaagtteccgtttt 304. aaggctcgcaatccgtcttTTTTTctgagtcaaagcat

Table 35: Blocking Label Extender of HPV type 59 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 59 305. ggeagtttgtatggtcgttgtgta 306. aatattcaatgttgtgcteaaatca 307. tacactataaataagtcattaaaagcaaat 308. gctgcatacggtgtacagtctcta 309. eataaaatgaaatgcatttcagacae 310. aatctctataatatcttaattctcttactcttg 311. cttttttcagttatatgctttaatttatc 312. tatatattccagctatattatggaatctt 313. gacacccacgacactgtectg 314. gatgattaaetccatctggttcatet 315. cagctegtetagctagtagcaaag 316. acaatgttgtgacgctgtggtt 317. ttgattattacacttacaacacacacac WO 2012/002964 [0063] In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from HPV type 68. In such embodiments, at least one Capture Extender probe (e.g.r at least 26 PCT/US2010/040649 WO 2012/002964 two, three, four, or five) comprises a target, hybridizing sequence selected from Table 36 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 318 to 324 (Tables 36). In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 37 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 325 to 338 (Table 37). The test may further employ a Blocking Label (BL), e.g„ at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 339 to 347 (Table 38).

Table 36: Capture Extender of HPV type 68 SEQ ID NO CAPTURE EXTENDER. OF HPV TYPE 68 318. etcatacaatataatetccaatetTTTTTctcttggaaagaaagt 319. tsttaagtscc^fftttttctacTTTTTctcttgeaaagaaagt 320. cgcttactgatccagcagtgcTTTTTctcttggaaagaaaat 321. cggtgggctttegtccatgTTTTTctcttggaaagaaagt 322. ataaetdaacacaatttertscaTTTTT etcttggaaagaaagt 323. cccacaacgcttctactactTTTTTctcttggaaagaaagt 324. caaaatttaataaelccataaacaacTTTTT etcttggaaagaaagt Table 37: Label Extender of HPV type 68 SEQ ID NO LABEL EXTENDER OF HPV TYPE 68 325. cttctecaataeacacaatetattataacTTTTTgaagttaccettlt 326. cat at acctctetecatt ata gtr gcTTTTTcigaatcaaagcat 327. attiaatacatgattggcatg.cagTTTTTgaagttaccgtttt 328. cslagttcccetattttascataaaTTTTTctgagtcaaagcat 329. gcatacaccaattcc aaataatat ϊ ΤΪ TI gaagttaccgtttt 330. ctttgtattagttatggtttctaatgtaattTTTTTctgagtcaaagcat 331. actcatecaccttatcaataaaUatataaTTTTTgaagttaocgtiit 332. taaacacaateatttcasa^TTTTTctgagtcaaagcat 333. caaetstatltcattatataaaeTTTTTaaaaiiacegtttt 334. tsctcgtgacatacaaggicaacTTTTTctgagtcaaagcat 335. ctatttcatcatctgaatctcctaatTTTTTgaagttaccatttt 336. aactecataatcaeettcatTTTTTctgagtcaaaacat 337. actettattcatcccatctaTTTTTgaagttaccatttt 338. caacacaaacactaaattctataacTTTTTctgagtcaaagcat 27 PCT/US2010/040649

Table 38: Blocking Label Extender of HPV type 68 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 68 339. ctacacataggtcactaaaggcaaatt 340. caaatggtaccccgtctctataca 341. gctattttatgtaatcttcgtttigt 342. cgacactgtcctgtaaagtttcct 343. gtatgcgtctgcggtcctct 344, catagttacttaaacttgtgtttcttgac 345. gctagtagtagatgttggtggtgatt 346. agttgc agtgc cttgttac actta 347. tgttgtagtgtecgeaggttgt WO 2012/002964 [0064| In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from low risk-HPV type 6. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 39 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 348 to 354 (Tables 39). In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 40 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 355 to 368 (Table 40). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 369 to 375 (Table 41).

Table 39: Capture Extender of HPV type 6 SEQ ID NO CAPTURE EXTENDER OF HPV ΓΥΡΕ 6 348. teeataeccacctcEiaaTTTTTctcttegaaagaaagt 349. cacecgcaegcigeataTTTTTctcttggaaagaaagt 350. tttttccatgaaattctaggcagTll'ITctcttggaaagaaagt 351. taggcagcgacccttecaTTTTT ctcttggaaagaaagt 352. caatatcctttagggtaacatgtcttcTTTTTctcttggaaagaaagt 353. cagaa get gtt gcacttctct gaTTTTTct e ttggaaagaaagt 354. ggtcttcggtgcgcagatgTTTTT c tcttggaaagaaagt 28 PCT/US2010/040649

Table 40: Label Extender of HPY type 6 SEQ ID NO LABEL EXTENDER OF HPV TYPE 6 355. attaatttseaacatatgcatagaiaTTTTTgaagttaecgtttt 356. gtgcattcttscaaaacacacaTTTTTctgagtcaaagcat 357. taiga ataaatctct act gtggtcaTTTTT gaagttacc gtttt 358. caggacctttaggtgtttatatgcaTTTTTctgagtcaaagcat 359. tcttcaactgttgttgcatatccaTTTTTgaagttaccgtttt 360. cacgtctaagatgtcttgtttagtttctTTTTTctgagtcaaagcat 361. cgccttggttagtetatgttttacctTTTTT gaagttaccgtttt 362. cgtacaatttagctttatgaaccgTTTTTctgagtcaaagcat 363. ggtctggaggttgcaggtctaataTTTTTgaagttaccgittt 364. tgctcatagcaatgtaa<x»tacagTTTTT etgagteaaagcat 365. acctcatcttctgagctgtctactaatTTTTTgaagttaccgtttt 366. cttgtccgtccacttcgtccTTTTTcteagtcaaaecat 367. cagtcgaacgttgctgtcacatTTTTTgaagttaccgtttt 368. tgtctgtttctgtacactgcacaacTTTTTctgagtcaaagcat

Table 41: Blocking Label Extender of HPY type 6 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 6 369. gcataatcaaagtgtctatattggttta 370, tgacacaggtagcaccgaattag 371. tttctacti.eaeaeageggtttg 372. catgeatgttgtecagcagtg 373. gaaatgttgttttaaaggttgtgaat 374. ccacagcaacaggtcactatttg 375. ggacacactatgtttagtgttcccaa WO 2012/002964 (0065} In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from low risk-HPV type 11. In such embodiments, at least one Capture Extender probe (e.g, at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 42 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist, of a sequence selected from SEQ ID NOS: 376 to 382 (Tables 42), In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 43 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 383 to 398 (Table 43). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 399 to 403 (Table 44). 29 WO 2012/002964 PCT/US2010/040649

Table 42: Capture Extender of HPV type 11 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 11 376. caaagaaagattaaacgtcttgcacTTTTT ctcttggaaagaaagt 377. cgcacigaatttgcagagtgigTTTTTctctiggaaagaaagt 378. tggtttcttcttctactgtaggtgcTTTTT ctcttggaaagaaagt 379. cgaatt aac acttttaaaatat c itcalTTTTT ctcttgga aagaaagt 380. agcgtgcctticccaatatgTTTTTetcttegaaagaaaal 381. accctacagggteaggaggctTTTTTctcttggaaagaaagt 382. SEQ ID NO gtgcgtc-ttgtttgtecacctTTTTTctcttggaaagaaagt Table 43: Label Extender of HPV type 11 LABEL EXTENDER OF HPV TYPE 11 383. tggaggcatctttactttccataaTTTTT gaagttaccgtttt 384. aactggtctatagatgttgcagacgTTTTT ctgagtcaaagcat 385. atatgcataratctctgcggtggTTTTTgaagttacegtttt 386. acacaacctttaggttcttataggcTTTTTctgagtcaaagcat 387. aaecaacaggcacacecteTTTTT gaagttaccgtttt 388. ggttaattttcccttgcagttctTTTTT ctgagtcaaagcat 389. Cg£Cltl2tg&amp;C&amp;C&amp;!22iB.3.C3.&amp;TTTTT £cl&amp;gtt&amp;CC2ti tl 390. tgctttagtttttctatttcacacaaTTTTTctgagtcaaagcat 391. cttccactggttatttagttttatgaTTTTT gaagttaccgtttt 1 392. ccagcagtgtaagcaacgaccTTTTT ctgagtcaaagcat 393. gtcttciaattgctcatagcaatgiaTTTTT gaagtiaccglttt 394. tgtccacctcatcttctgagctTTTTTctgagtcaaagcat 395. gtatttggtaatgttgtgttaaaggtt'm'Tr gaagttaccgtttt 396. cacatccaragcaacaggicaTTTTTctgagtcaaagcat 397. caaccagtcggacgttgctgtTTTTT gaagttaccgtttt 398. atgtctccgtctgtgcactccaTTTTTctgagtcaaagcat Table 44: Blocking Label Extender of HP V type 11 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 11 399. tcagtgcattcctgcaaaaca 400. caaagggaaagttgtctcgec 401. atatgcagcaiaattaaagtgtctatatt 402. 403. taacaagtcttccatgcatgttgt gcaggtctagtactatatcctttaggg |0066] In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from low risk-HPV type 40. In such embodiments, at least one Capture Extender probe (e.g., 30 PCT/US2010/040649 WO 2012/002964 at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 45 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 404 to 410 (Tables 45). In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 46 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 411 to 424 (Table 46). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 425 to 432 (Table 47).

Table 45: Capture Extender of HPV type 40 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 40 404. tcatacageatcciggccta'TTTTTctcttgaaaagaaaat 405. ggaccgtcttgcaaaacacacTTTTTct.cttggaaagaaagt 406. cgtggacatgcagcgtgtTTTTTctcttggaaagaaagt 407. aaagaattgcgtciictttacaatatTTTTTctcttggaaagaaagt 408. agtttaaacatacagettcagaataTTTTTctcttggaaagaaagi 409. acacceasttaclactttaaatEattgTTTTTctettggaaagaaagt 410. tgatggaacaatgcactactaagTTTTTcicltggaaagaaaEt Table 46: Label Extender of HPV type 40 SEQ ID NO 411. LABEL EXTENDER OF HPV TYPE 40 tgtaatattgcactaatcacacaatTTTTTgaagttaccgtttt 412. aateaatttgcaacgtaggcaaTTTTTctgagtcaaagcat 413. ggccastacctcagctstttttaTTTTTgaagttaccgtttt 414. cacaacatataactctctaaaggcaaaTTTTTctgagtcaaagcat 415. ccgtecaggtccaggcacTTTTTgaagttaccgtttt 416. atctaaagtttctatattgattoc^TTTTT ctgagtcaaagcat 417. gttaatcctatctcttcttccacaTTTTT gaagttacegtttt 418. cagcatclaatccttacttataaaataTTTTT ctgagtcaaagcat 419. cccMccacaaatcttttaatttTTTTTgaagttaccetttt 420. catttcttccagcaalataaacagtaTITlTctgagtcaaagcat 421. 422. gagctgtctaattgctcgttacTTTTTgaagttaccgtttt ct gtt catest catcttctaa etctTTTTT ctgagtcaaagcat 423. tctactgtataagctetctaattaatcTTTTTgaagttaccgtttt 424. cataggttgctcacectcTTTTTctgagtcaaagcat 31 PCT/US2010/040649

Table 47: Blocking Label Extender of HPV type 40 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 40 425. ggaaagtcgtcgcgcca 426. gttggtgcataggctgcgi 427. gacaaaggcttgtggcacttg 428. ggttggtttttt ccacggga 429. gcgttggcctttctccatg 430. caggtttaacacaatgtctccga 431. caaatttacttgcaggtcctgttg 432. cgcaccaaacactgacaaaatac WO 2012/002964 [0067] In. certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from low risk-HPV type 42. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 48 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 433 to 439 (Tables 48), In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in fable 49 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 440 to 451 (Table 49). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 452 to 462 (Table 50).

Table 48: Capture Extender of HPV type 42 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 42 433. acattctgccttaactgggaecTTTTT ctcttggaaagaaagt 434. cctsttaagtfictttttacaccTnTTctcttggaaagaaagt 435. acgceagcacctctscsTTTTTctcttggaaagaaagt 436. caataiaaattgaaatcttgiacctgtatcTTTTTctcttggaaagaaagt 437. cattgtcctctscaatscataTTTTTctcttggaaagaaagt 438. cgctatatstcctatttgsctTTTTTctcttggaaagaaagt 439. cttatgtccgcctctgtacactgTTTTTctcttggaaagaaagt 32 PCT/US2010/040649

Table 49: Label Extender of HPV type 42 SEQ ID NO LABEL EXTENDER OF HPV TYPE 42 440. 441. ctetgatgaeacasatataccteTTTTTgaagttaccgtttt tacacaattggtataatgtacgteeTTTTTcteagtcaaaecat 442. gcaatgtcagcccaaattcctTTTTT gaagttac cgtttt 443. 3.ci3,tSCciS2ii£liitCtSt3.3,3,ttCCTTTTTci.g!igtC33.3gC3l 444. caaaatgctgatctttcgtagtstTTTTTgaagttaccgtttt 445. agtccagtttetttctccactgtatacTTTTTctgagtcaaagcat 446. gataacggcttttgaeacaaggTTTTTgaagttacegtttt 447. aaMgatggtttttttcactotgtTTTTTctgagtcaaagcat 448. atgtcaaacaaaacaatatcctttaTTTTTgaagttaccgtttt 449. atesetgtctcacacsttestTTTTT ctgagtcaaagcat 450. caaaaacatetgttgcaegtttTTTTTgaagttaccgtttt 451. Table 50: Blocking Label Extender of HPV type 42 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 42 452. 45*)* caccac taccaaat c ttta aa atggt gC&amp;tc5t&amp;’g&amp;&amp;&amp;j^tCCitCCtCOc5 454. attctaaacaaaatgcacatgca 455. cgcagtgcacaaattttagaattaa 456. cacatctaatttgttgttcttetaaaagt 457. caccgacccgtccactgaca 458. gggtaggcgtctetecacg 459. caattgttcatagcaatacaggtca 460. tggteatcticatetgagctgte 461. 462. cacaacgagtttaacagacttgtaaca WO 2012/002964 [00681 in certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from low risk-HPV type 43, In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 51 (underlined), At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 463 to 468 (Tables 51). In such embodiments, at least one Label Extender (e.g,, at least two, three, four, or five) may comprise a target-hybridizing sequence shown in fable 52 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 469 to 482 (Table 52). The test may further employ a Blocking Label (BL), 33 PCT/US2010/040649 WO 2012/002964 e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 483 to 488 (Table 53).

Table 51: Capture Extender of HPV type 43 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 43 463. agcatgcagcaaacggatate'IT IT T ctcttggaaagaaagt 464. ccateaaartgtagacaesecaTTTTTctcttggaaagaaagt 465. tcaaastscctatatteacttatttti 11 j ] j ctcttggaaagaaagt 466. acagtatctgcatatgctgcgtagTTTTTctcttggaaagaaagt 467. atgcatgatttccagcaatatagTTTTTctcitggaaagaaagt 468. ttaatgtgcccaacagcaggTTTTT ctcttggaaagaaagt Table 52: Label Extender of HPV type 43 SEQ ID NO LABEL EXTENDER OF HPV TYPE 43 469. catcacacaactcaaatatagtccgTTTTTgaagttaccgtttt 470. acagagtagacaaagttatgttacaciTTTTTctgagtcaaagcat 471. acttccgtggtaagtaaccactteTTTTTgaagttaecgtttt 472. ttiaaatctctaaatgcaaacsataatTTITTctgagtcaaagcat 473. aacactwtttgcttagtttcttcttcffT'rTTgaagttacegtttt 474. cttacagcatctaatgcacaaatcaTTTTTctgagtcaaagcat 475. tggtgataatggcttgtggcaTnTTgaagttaccgtttt 476. gcacaatatcctgtacttittccac'TTTTTctgagtcaaagcat 477. atgtatittaaa saattgtgccttttTTTTT gaagttaccgtitt 478. gcagtatcctttccacacgctTTTTTctgagtcaaagcai 479. tggttgcatagttagcacatagtctTTTTTgaagttaccgtttt 480. cattacaggttaagcttctagettcTTTTTctgagtcaaagcat 481. attacacacagacaggatgtgcacTTrTTgaagttaccgtttt 482. aaagcactgcacaacaagtceaTTTTTctgagtcaaagcat Table 53: Blocking Label Extender of HPV type 43 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 43 483. cigaicgtcggettttttcc 484. tcigagtctgagetgtctaattgct 485. gggitgctcacgctcatcc 486. acttgctggtcctgttgcgt 487. tctgttacaactctgtaaacttgtagattc 488. tcitctagcttcttgatgtcactgtc [0069) In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from low ri&amp;k-HPV type 44. In such embodiments, at least one Capture Extender probe (e.g.. at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 34 PCT/US2010/040649 WO 2012/002964 54 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 489 to 494 (Fables 54). in such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Table 55 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 495 to 506 (Table 55). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 507 to 5.10 (Table 56).

Table 54: Capture Extender of HPV type 44 SEQ ID NO CAPTURE EXTENDER OF HPV TYPE 44 489. cctagcasfacttaacsrtttttctg'n ΤΊ Tctcttgqaaagaaagt 490. acacatccasaattsacttatttatTTTTTctcttggaaagaaagt 491. tttaatgaatcgcgccttgtcTTTTTctcttgsaaagaaagt 492. cgacccttccaaatatcttgtaaTTTTTctcttggaaagaaagt 493, cctttaaaataatataatttccatacaTTTTTctcttggaaagaaagt 494. gaaattccagctgtaaaacaatttTTTTTctcttggaaagaaagt Table 55: Label Extender of HPV type 44 SEQ ID NO LABEL EXTENDER OF HPV TYPE 44 495. 496, gcagaegtegaggcatttgcTTTTTgaagttaccgtttt ccttgcacaactggtctatacmgtTTTTTctgagtcaaagcat 497. aiigtgcataggaatgttgcaci 11' IT rgaagiiaccgtttt 498. caaaacacgcataaaatitgcagTTTTTcigagtcaaagcat 499. acaaatggcacaggctgcaTTTTTgaagttaccgtttt 500. tgatig.accitacettgtMtictacTTTTTctgagtcaaagcat 501. ccgcgtagttaaaatgcetaaatTTTTTgaagttaccgtttt 502. ttcttcttccactgttaet gcatatcTTTTT ctgagtcaaagcat 503. ggcacaaataacagcgtatcaTTTTTgaagttaccgtttt 504. cacgtggcacaatagtttgtTTTTTctgagtcaaagcat 505. caatgtaggcctacaggstcaaTTTTTgaagttaccgtttt 506. tctgagetgtctaattgctoatteTTTTTctgagtcaaagcat Table 56: Blocking Label Extender of HPV type 44 SEQ ID NO BLOCKING LABEL EXTENDER OF HPV TYPE 44 507. etacatataactgtttatatgcgaatgaataaa 508. aatggaaagtttcc tcggtaca 509. caatatgtggcgcaccttttc 510. tgatgtccaacaatggaagcag PCT/US2010/040649 WO 2012/002964 [0070] In other embodiments, the target polynucleotide is bepadnavirus polynucleotide, such as HBV. In such embodiments, the target polynucleotide is one or more polynucleotide sequences encoding the viral core protein, HBcAg; the proteolytic processing protein, HBeAg, anchor the viral surface antigen, HBsAg. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence as selected from Table 57 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 511 to 519 (Tables 57). At least one label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 58 (underlined). At least one Label Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 520 to 535 (Table 58). The test may optionally employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 536 to 553 (Table 59).

Table 57: Capture Extender of HBV SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF HBV 511. jassgaRWccgcgt^agagatttttctcttggaaagaaagt 512. tgcRacgtgcagaggtgaatttttctcttggaaagaaagt 513. RastccaasaetcetcttatgYaagactttttctcttggaaagaaagt 514. cagagstsaaaaastt2cat2R.tttttctcttggaaagaaagt 515. aMggRte aatgtccatgcc tttttctc ttggaaa gaaagt 516. tcaeaaeecaaaaaMgagagtaacttttttctcttggaaagaaagt 517. atDgcttgcctKagtgcHgtatgtttttctcttggaaagaaagt 518. cggaagtgttgataagataggggtttttctcttggaaagaaagt 519. ctKcetctecgaggcgaatttttctcttggaaagaaagt

Table 58: Label Extender of HBV SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HBV 520. ggcagatgagaaggcacaeactttttgaagttaccgtttt 521. gcgaagtgcacacggWcctttttctgagtcaaagcat 522. tcggtcgttgacattRcWgRtttttgaagttaccglttt 523. cagtctttgaagtaKgcctcaaggtttttctgagtcaaagcat 524. gcctacagcctccYaRtacaaagactttttgaagttaccgtttt 525. teMt££teMR.caSaccaattiattitttctgagtcaaagcat 526. ggacateWacaWgagatgatYa ggtttttgaagttaccgtttt 527. cagcttggaggcttgaacagtRtttttctgagtcaaagcat 528. agactctaaggc YtcY caatactttttgaagttaccgtttt 529. RtgaggtgaRcaatgYtcMagtttttctgagtcaaagcat 530. cctcKtcatctaacaacagtagtYtctttttgaagttaccgtttt 36 PCT/US2010/040649 SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HBV 531. caacttaaaaacttaaacaetRtttttctgagtcaaagcat 532. ceaca'CSS'CsattaaeaYtttttgaagttaccgtttt 533, attccceagatteaeatctYctgtttttctgagtcaaagcat 534. aWatccaaaaRatactaacattaaatttttgaagttaccgtttt 535. cccMataaaRtttcccaccttattttttctgagtcaaagcat

Table 59: Blocking Label Extender of HBV SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE I SEQUENCE OF HBV 536. gcgttcacggtggtctcca 537. cttgggcaRgWHB Y S DYgg 538. aRctcctcccaStcHKtaaaY aMa 539. ctoJR.tCtCCtcCCCC 540. cYaaagccaYccaaggca 541. ccacagWagctccaaattctttat 542. gDagatcHcKDaYDgaaggaaagaag 543. agagcWgaggcKgtRtcDa 544. Y atY aRBtcMccccaRcaV agR 545. ccacccaggtRgM Y aRaKt 546. tg Y WggRtcttccaaattaH Y W c 547. cataR YtKactactaRDtccctRga 548. WYtttaRRcccatRttaRYRttRa 549. aW atRtgaaaccacaatagttgY ctRa 550. gtYtctctYccaaaagtRagRcaRg 551. cRaaagaSaccaaataYtcWaKDacH 552. gWggagtgcgaatccacactc 553. cattW ggtggtct RtaDgcDg WO 2012/002964 10071] The invention also provides a method for detecting a viral target polynucleotide from a RNA virus such as HIV or paramyxovirus (flu virus). The target polynucleotides for detecting a HIV infection can be HIV-A, -B or -F. in such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence of Table 60 (underlined). At least one Capture Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 554 to 558 (Table 60). At least one Label Extender (eg., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from T able 61 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 559 to 567 {Table 61). The test may optionally including a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 568 to 571 (Table 62). 37 PCT/US2010/040649

Table 60: Capture Extender of HIV-A SEQ ID NO CAPTURE. EXTENDER NUCLEOTIDE SEQUENCE OF HIV-A 554. ttcatitcctccaatccccttatntnlTITTctcttggaaagaaagt 555. attecteteatatctttcatgttcttcttgaT'TiTTctcttggaaagaaagt 556. actectaccagaattactttcccttctaaatgTTTTTctcttggaaagaaagt 557. cectggtaaaattgctgccattgtctTTTTTctcttggaaagaaagt 558. ctecttgactttggggaltgtagggaTTTTT ctcttggaaagaaagt

Table 61: Label Extender of HIV-A SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HIV-A 559. ctgattceagagctga ctaalttatctac ttgTTTTT ctgagtcaaageatgaagttae 560. gccttatctatcccatctaaaaaiagtalcttcTTTTTctgagtcaaagcatgaagttac 561. agaltaaaatcactagccattgctctccaTTTTTctgagtcaaagcatgaagttac 562. ggctactaittccttigctactatasstggcTTTTTctgagteaaagcatgaagttac 563. aaetacagtciaectstecatacatascTTnTctgagtcaaageaigaagttac 564. ctacttctatatagccactsactacatas TTTΎTctgagtcaaagcatgaagttac 565. cctgccctgttictgctggaaiaactiTTTTTeigagtcaaagcatgaagttac 566. atatetactacitttacteaecatectceTTTTTctgagicaaagcatgaagttac 567. ecaceaacaggctgctitaaatgcagTTTTTctgagtcaaagcatgaagttac

Table 62: Blocking Label Extender of HIV-A SEQ ID NO BLOCKING L ABEL EXTENDER NUCLEOTIDE SEQUENCE OF HIV-A 568. ttccccttttagttgacatttatcacagct 569. igtgcaatctaattgccacatccctg 570. tgctaattttagcaaaaagtatgctgcct 571. atcccaaattcctgttggatgtttgc WO 2012/002964 [0072] In another embodiment, the target polynucleotide is an. HIV subtype B (HIV-B) polynucleotide. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target-hybridizing sequence selected from Table 63 (underlined). At least one Capture Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 572 to 577 (Table 63). At least one Label Extender probe (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 64 (underlined). At least one Label Extender may comprise, consist, essentially of or consist of a sequence selected from SEQ ID NOS: 578 to 590 (Table 64). The test may optionally employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 591 to 595 (Table 65). 38 PCT/US2010/040649

Table 63: Capture Extender of HIV-B SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF HIV-B 572. tecttctatatacccactsectacateaactTTTTTctcttggaaagaaagt 573. caacaeeceecctteaccscTTTTTctcttggaaagaaagt 574. tcetscttaacccccecccacTTTTTctcttggaaagaaagt 575. acactataetccccaatcccccctcTTTTTctcttggaaagaaagt 576. 577. SEQ ID NO ctttccaeaggagettiectggtccTTTTTctcttggaaagaaagt actcttatcttgtattactactsccccltcacTTTTTctcttggaaagaaagt Table 64: Label Extender of HIV-B LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HIV-B 578. caatctaetteccatattccteeactacastTTTTTctgagtcaaagcatgaagttac 579. gctaccaeeataactettcettctaaatgtetaTTITTctgagtcaaagcatgaagttac 580. accctstetctacteesatcacttcTTTTTctgagtcaaagcatgaagttac 581. tgtatgtaUgtctttactggccatcttccTTTTTctgagicaaagcatgaagttac 582. ttctttattcaiagattctactactccltgactTTTTTctgagtcaaageatgaagttac 583. gatctcttacctgtcctataatttictttaaTTTTTctgagtcaaagcatgaagttac 584. tttgtactgctatcttaasatgttcagcctTTTTTctgagtcaaagcatgaagttac 585. ttcttttaaaattetggatgaaiaeteccaTTTTTctgagtcaaagcatgaagttae 586. ctgttgctattatgtc.tattattctctcccctTTTTTctgagtcaaagcatgaagtiac 587. aatttgtttttgtaattctttggtttgtatgtTTTTTctgagtcaaagcatgaagtiac 588. tstaataeaccegaaaatttteaatttttgiTTTTTctgagtcaaagcatgaagttac 589. accatctgttttccataatccctaatgatTTTTTctgagtcaaagcatgaagttac 590. cctetctactteceaiacaatcatcaectTTTTTctgagtcaaageaigaagttac Table 65: Blocking Label Extender of HIV-B SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HIV-B 591. 592. tgctaattttaagagaaagtatgctgtttcct actactggtgaagttgctgceattgtc 593. ttggggattgtagggaatgccaaat 594. tttccaaagtggatctctgctgtccc 595. ctttacttttcttcttggtactactttiatgtc WO 2012/002964 [0073] In a further embodiment, the target polynucleotide may be an HIV subtype F (HIV-F) polynucleotide. In such embodiments, at least one Capture Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 67 (underlined). At least one Capture Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 596 to 600 (Table 67). At least one Label Extender ( e.g., at least one, two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 68 (underlined). At least one Label Extender may comprise, 39 PCT/U S2010/040649 WO 2012/002964 consist essentially of, or consist of a sequence selected from SEQ ID NOS: 601 to 608 (Table 68), The test may optionally employ a Blocking Label (BL), e.g., containing the sequence of SEQ ID NO: 609 (Table 69).

Table 66: Capture Extender of HIV-F SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF HIV-F 596. ttctttagctctttattcattgattctactactccTTTTTctcttggaaagaaagt 597. ttcccctecactgtaccccccaatcTTTTTctcttggaaagaaagt 598. actgtccctgtaataaacccggaaattttTTTTTctcttggaaagaaagt 599. taccccttcacctttccagagtagctTTTTTctcttggaaagaaagt 600. cetcacetgccatctgttttecataatcTTTTTctcttggaaagaaagt

Table 67: Label Extender of HIV-F SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HIV-F 601. ttcagettgatctcttacctgtcctatgalcTTTTTctgagtcaaagcatgaagUac 602. atactgceatttgtactgctgtcttaagatgTTTTTcigagtcaaagcatgaagttac 603. cccccttttcttttaaaattgtggatgaTTTTTctgagtcaaagcatgaagttac 604. ttgtatgtctgttgctattalgtctaltattctTTTTT ctgagtcaaagcatgaagttac 605. gaattttlgtaacttgtttttgtaattctttagtTTTTTctgagtcaaagcatgaagtta 606. ttactaetcctttccaaactggstctctTTTTTctgagtcaaagcatgaagttac 607. ctacttttatttcactattgtcttgtatgactacTTTTTctgagtcaaagcatgaagtta 608, tcatcetgtctacctgccaeacaaiTTTTTctgagteaaageatgaagttac

Table 68: Blocking Label Extender of HIV-F SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HIV-F 609. cctaatgatctttgcttttcttcttggca [0074] The target polynucleotide may be a polynucleotide of a respiratory viruses, such as a influenza H1N1. For example, the target polynucleotide may be a sequence encoding a portion of hemaglutinin (HA), neuraminidase (ΝΑ), M protein, F protein, N protein, G protein, etc. In an exemplary embodiment, the target polynucleotide is an HA sequence. In such embodiments, at least one Capture Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 69 (underlined). At least one Capture Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 610 to 629 (fables 69). At least one Label Extender (e.g., at least two, three, four, or five) may comprise a target hybridizing sequence selected from Table 70 (underlined). At least one Label Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 630 to 689 (Table 70). The test may optionally 40 PCT/US2010/040649 WO 2012/002964 employ a Blocking Label (BL), e.g., at least one, two, or three BLs each, containing a sequence selected from SEQ ID NOS: 690 to 707 (Table 71).

Table 69: Capture Extender of Flu Virus, MINI SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF Flu Vims, H3N1 610. 611. RgUctanggaaaatgaaagaactttTTTTTcicttggaaagaaagi caaaateaecaeeestcaseTTTTT ctcttggaaagaaagt 612. tgccggtttcattgaagggTTTTT ctcttggaaagaaagt 613. ccagcctcccatttcagaatatacTTTTT ctcttggaaagaaagt 614. acacccaagggtgctataaacaTTTTT ctcttggaaagaaagt 615. caagccggaaatagcaataagaTTTTTctettggaaagaaagt 616. aaaaggaaattcatacccaaagcTTTTT ctcttggaaagaaagt 617. aaaggtgtaacggcagcatatTTTTT ctcttggaaagaaagt 618. ctcaatatcatcatttsaaagatttTTTTTctcttggaaagaaagt 619. 620. ggttctattagaaaatgaaagaactttTTTTTctcttggaaagaaagt 621. caaaatgagcaggggtcaggTTTTTctcttggaaagaaagt 622. tgccggtttcattgaagggTTTTT ctcttggaaagaaagt 623. ccagcctcccatttcagaatatacTTTTT ctcttggaaagaaagt 624. acacccaagggtgctataaacaTTTTTctcttggaaagaaagt 625. 626. caasccscraaataacaataasaTTTTTctettggaaagaaagt aaaaggaaattcatacccaaagcTTTTTctcttggaaagaaagt 627. aaaggtgtaacggcagcatgtTTTTT ctcttggaaagaaagt 628. ctcagtgtcatcatttgaaaegtttTTTTIVtcttggaaagaaagt 629. ccagtaaactgattaactggttcttcaTTTTT ctcttggaaagaaagt Table 70: Label Extender of Flu Virus, HI N1 SEQ ID NO LA BEL EXTENDER NUCLEOTIDE SEQUENCE OF Flu Virus, H1N1 630. tggaettacaatgcegaactgttTTTTTgaagttaccgttil 631. tgatgatggtttcctggacattTTTTTctgagtcaaagcat 632. ggtaaagaattcaaccacctggaT'nTTgaagttaccgtitt 633. aagatgaataeacagttcacagcastaTTTTTetgagtcaaagcat 634. cacacaeaatgccattgaceagTiTTTgaagttaccgtttt 635. atatecaecegaccteaaeaeTTTTTctgagteaaagcat 636. tggatggtacgettatcaccalTTTTTgaagttaccgtttt 637. gggteeacagggatggtaeaTTT'Trctgagtcaaagcat 638. aagcctatttgggaccatTTTTTgaagttaccgtltt 639. ggaatatcccgtctattcaatctagTTTTTctgagtcaaagcat 640. tga gactggccacaggattgaT 1 ϊ ϊ I gaagttaccgtttt 641. iccaaaaiatgtaaaaagcacaaaatTTTTTetgagtcaaagcat 642. cacgattgcaatacaacttgtcaaTTTTTgaagttaccgtttt 643. ggtattatcatttcagatacaecagtcTTTTTetgagtcaaagcat 644. gtegtaccgagatatgeattcgTTTTTgaagtlaccgUtt 41 PCT/US2010/040649 WO 2012/002964 SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Flu Virus, H1N1 645. cattceaaecaactesaaatctaTTTTTctgagtcaaagcat 646. agtagaecceaeaaacaaaataaTTTTTgaagttaccgtttt 647. gggagaatgaaciattactggacactTTTTTctgagicaaagcat 648. aatacaaatacatatatitttsissTTTTTgaagttaccgtttt 649. tgctgaccaacaaagtctctatcagTTTTT ctgagtcaaagcat 650. ttctacaaaaatttaatatggctagttaaTTTTT gaagttaccgtttt 651. ectcatsctggagcaaaaaaeTTTTTctgagteaaagcat 652. ggcccaatcatgactcgaacTTTTTgaagttaccgtttt 653. gagatattccccaagacaagttcatTTTTTctgagtcaaagcat 654. tgaggagctaagagagcaattgagTTTTTgaagttaccgtttt 655. tacccaggagatttcatcgallaTTTTTctgagtcaaagcat 656. cciaattcaaacaataaaacatetTTTTTgaagttaccgtttt 657. catggtcctacattgtggaaacaTTTTTctgagtcaaagcat 658. tsaatcactctceacaacaasctTTTTTgaagttacegtttt 659. ggatcctgggaaaiccagagtaTTTTT ctgagtcaaagcat 660. tggacttacaatgccgaactettTTTTT gaagttaccgtttt 661. tgatsatgglttcctggacattTTITIxtgagtcaaagcat 662. ggtaaagagttcaaccacctggaTTTTT gaagttaccgtttt 663, aagatgaatacacagttcacaecagtaTTTTTctgagicaaagcat 664. cacacagaatgccattsacgagTTTTTgaagttaccgtttt 665. atatgca gcc gacctaaagagTTTTTctga gtcaaa gcat 666. tggatggtacggttatcaccatTTTTTgaagttaccgtitt 667. gggtggacagggatggtagaTTTTTctgagtcaaagcat 668, aggcctatttggggccatTTTTT gaagttaccgtttt 669. ggaatatccc gtctattcaatctagTTTTT ctgagtcaaagcat 670. tgagactggccacaggattgaTTTTT gaagttaccgtttt 671. tccaaaatatgtaaaaagcacaaaatTTTTT ctgagtcaaagcat 672. cacgaitgcaaiacaacttgtcaaTTTTTgaagttaccgtttt 673, sgtattaicatttcagaiaeaccagteTTTTTctgagteaaagcat 674. gtsetacceasatateeattceTTTTTgaagitaecgittt 675. cattcgaagcaactggaaatctaTTTTT ctgagtcaaagcat 676. agtagaeccgggagacaaaataaTTTTT gaagttaccgtttt 677. ggeagaatgaactattacteeacactTTTTTctgagtcaaagcat 678. aatgcagatacatatgtttttgtggTTTTTgaagttaccgtttt 679. tgctgaccaacaaagtctctMcagTTTTT ctgagtcaaagcat 680. ttctacaaaaatttaatatagctagttaaTTTTTgaagttaccgtttt 681. ccicatgciggagcaaaaagcTTTTTctgagtcaaagcat 682. ggcceaateatsaetceaacTTTTTgaagttaecgtttt 683. gagatattccccaagacaagttcatTTTTTctgagtcaaagcat 684, tgaggagctaagagagcaattgagTTTTT gaagttaccgtttt 685. tacccageagatttcatcaattaTTTTTctgagtcaaagcat 686. cctagttcagacaatggaacgtgtTTTTT gaagttaccgtttt 687. catggtcctacattgtggaaacaTTTTTctgagtcaaagcat 688. tgaatcactctccacagcaaactTTTTTgaagttaccgtttt 689. ggatcctgggaaatccagagtgTTTTT ctgagtcaaagcat 42 PCT/US2010/040649

Table 71: Blocking Label Extender of Flu Virus, H1N1 SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Flu Virus, mm 690. aaaaagaatagagaatttaaataaaaaagt 691. attactaacaaagtaaattctgttattgaa 692. atccgatcacaattggaaaatg 693. caatggaaagaaatgctggatct 694. cccaaagtgagggaicaagaa 695. ggtcatcaagatacagcaagaagtt 696. gggcattcaccatccatctactag 697. gggaaagaagtcctcgtgctatg 698. tcagcaaatcctacaUaatga iaaa 699. aaaaagaatagagaatttaaataaaaaagt 700. attactaacaaagtaaattctgttattgaa 701. atcegatcacaattggaaaatg 702. caatggaaagaaatgctggatct 703. cccaaagtgagggatcaagaa 704. ggtcatcaagatacagcaagaagtt 705. gggcattcaccatccatctactag 706. gggaaagaagtcctcgtgctatg 707. tcagcaaatcctacattaatgataaa WO 2012/002964 [0075] The present invention further provides a method for detecting viral target polynucleotides from a flavirus, for example, HCV. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target-hybridizing sequence selected from Table 72 (underlined). At least one Capture Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ 1D NOS: 708 to 714 (Tables 72). At least one Label Extender (LE) (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 73 (underlined). At least one Label Extender may comprise, consist essentially of, or may consist of a sequence selected from SEQ ID NOS: 715 to 727 (Table 73). The test may optionally employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 728 to 732 (Table 74).

Table 72: Capture Extender of HC V SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF HCV 708. laacaccateectaeacactttctacaieaTTTTTctctiggaaagaaagt 709. aceaatteatccaaaaaaaaaccceatcTTTTTctcitggaaagaaagt 710. agcacacccaaatctccaaacattgaTTTTTctcttagaaagaaaat 711. aaacctGccaaegcactcgcaaTTTTTctcttggaaaaaaagt 712. cctaccetcaeectaacaaaccTTTTTctcttggaaagaaagt 43 PCT/U S2010/040649 SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF HCV 713. caccccaaeccctcatteccatagaeTTTTTctcttggaaagaaagt 714. cgaecseaatgtaccccateaeatcggTTTTTctcttggaaagaaagt Table 73: Label Extender of HCV SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HCV 715. ggtcctggaggcigca.eeacactcatacTTTTTctgagtcaaagcatgaagttac 716. ccecaeaccactatgectcicccgTTTTT ctgagtcaaagcatgaagttac 717. gtcctggcaattccggtgtactcaccgTTTTT ctgagtcaaagcatgaagttac 718. caacactactcggctagcagtctcgcggg'TTTTTctgagtcaaagcatgaagttac 719. caccctatcaggcagtaccacaaggccttTTTTTctgagtcaaagcatgaagttac 720. ttcgtgctcatggtgcacggtctacgTTTTTctgagtcaaagcatgaagttac 721. ggtgttac gtttgglttttctttgaggttta gTTTTT etgagtcaaagca Igaagttac 722. cccctgceceecaacaggtaaactccacTTTTTctgagtcaaagcatgaagttac 723. gtc gcgcgcacacccaacctggTTTTTetgagtcaaagca Igaagttac 724. 725. ttegggataggttgicgccticcaceaTTTTTctgagtcaaagcatgaagttac acggggtgacaggagccaicctgccTTTTTc-tgagtcaaagcatgaagttac 726. ceaaattgcgeaacetacgccggggTTTTTctgagtcaaagcatgaagUac 727. aagccgcacgtgagggtatcgatgaccttacTTTTTctgagtcaaagcatgaagttac Table 74: Blocking Label Extender of HCV SEQ ID NO 728. BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF HCV acttgacgtcctgtgggcggcgg 729. cgacgatetgaccaccgcccggga 730. ggttgcgaccgctcggaagtcttccta 731. ccaggggtacccgggctgagccca 7^9 tggggccccaactaggccgagagcc WO 2012/002964 [0076] The present invention further provides a method for detecting viral target polynucleotides from a coronavirus, for example, severe acute respiratory syndrome (SARS) virus. In such embodiments, at least one Capture Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 75 (underlined). At least one Capture Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 733 to 738 (Table 75). At least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 76 (underlined). At least one Label Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 739 to 750 (Table 76). The test may optionally employ a Blocking Label (BL), e.g., containing the sequence of SEQ ID NO: 751 (Table 77). 44 PCT/US2010/040649

Table 75: Capture Extender of SARS Virus SEQ ID NO CAPTURE. EXTENDER NUCLEOTIDE SEQUENCE OF SARS Vims 733. ccaetaaacteattaacteattcttcaTTTTTctettggaaagaaagt 734. etetatettascaecatttac-aatcaTTTTTctcttggaaagaaagt 735. ccttttacatencaccattgTTTTTctcttggaaagaaagt 736. gcaasattatetccagaaancaaaTTTTTctcttggaaagaaagt '•t'N "7 / J / . ecteccttaaeaaecteeatetTTTTTctcitggaaagaaagt 738. setttaecaccaaatatsccteTTTTTctcttggaaagaaagt

Table 76: Label Extender of SARS Virus SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF SARS Virus 739. caatctctaattectcaetafitatcatTTTTTgaagttaccgtttt 740. ggtgtaggttctggttctggctTTTTTctgagtcaaagcat 741. ggcaacattetcagtaagttttaaataaTTTTTgaagttaccgtttt 742 tccttaacgatgtcaacacatttaatTTnT ctgagtcaaagcat 743. gctacac caccacca tgtttcagTTTTT gaagttaccgtttt 744. gttgccttgUgaatgcacctTTTTTctgagtcaaagcat 745. ccatttaecttaatgtaatcatcactctTTTTTgaagttaecgtttt 746. caaeaccctcctactetaaeagggTniTctgagtcaaagcat 747. ccaacaacatgcagacacttcttaTTTTT gaagttaccgtttt 748. cctcacctgcatttaggttaggtTTTrrrrctgagtcaaagcat 749. tgtcctgteaattgaaattttcatatTTTTTgaagttaccgtttt 750. ctsacaacaatestecaastaa gaTTTTTctgagtcaaagcat

Table 77: Blocking Label Extender of SARS Virus SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF SARS Virus 751. ccalaggat tagcac tttgtgc c WO 2012/002964 [0077| in one aspect of the invention, the method provides for detection of bacterial target polynucleotides. Non-limiting examples of bacteria are: B. pertussis; Leptospira Pomona; S. paratyphi A and B; C. diphtheriae, C. tetani, C. botulinum, C. perfringens, C. feseri and other gas gangrene bacteria; B. anthracis; P. pestis; P. multocida; Neisseria meningitides; N gonorrheas; Hemophilus influenzae; Actinomyces {eg., Norcardia); Acinetobacter; Baciliaceae (e.g., Bacillus anthrasis); Bacteroides (e.g., Bacieroides firagflis); Blastomycosis; Bordetella (Bordetelia pertusi); Borrelia (e.g., Borrelia burgdorferi); Brucella; Campylobacter; Chlamydia; Coccidioides; Corynebacterium (e.g.,

Corynebacterium diptheriae); E. coli {eg., Enterotoxigenic E. coli and Enterohemorrhagic E. coli); Enterobacter (e.g. Enterobacter aerogenes); Enterobacteriaceae (Klebsiella, Salmonella (eg.. Salmonella typhi, Salmonella enteritidis, Serratia, Yersinia, Shigella); PCT/U S2010/040649 WO 2012/002964

Erysipelothrix; Haemophilus (e.g., Haemophilus influenza type B); Helicobacter; Legionella (e.g., Legionella pneumophila); Leptospira; Listeria (e.g., Listeria monocytogenes); Mycoplasma; Mycobacterium (e.g., Mycobacterium leprae, Mycobacterium tuberculosis and Mycobacterium bovis); Vibrio (e.g., Vibrio cholerae); Pasteurellacea (Pasteurella haemolytica): Proteus; Pseudomonas (e.g., Pseudomonas aeruginosa); Ricketisiaeeae; Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.); Shigella spp.; Meningiococcus; Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci); Ureaplasmas; Treponema pollidum; and Staphylococcus (Staphylococcus aureus and Staphylococcus epidermidis).

[0078] The target polynucleotides from the bacteria can be a polynucleotide, DNA or a RNA (such as a ribosomai RNA) that is involved in the replication and transcription of the bacteria, housekeeping genes, repetitive sequences, terminal inverted repeat sequence (TIR), miniature inverted repeat transposable element (MITE), CpGs, and/or polynucleotide encoding other bacterial proteins that are expressed on the cell surface or as integral membrane proteins.

[0079] In one embodiment, the present invention provides a method for detecting mycobacterium. The target polynucleotide for detecting mycobacterium infection can be a 16S RNA sequence. At least one Capture Extender (e.g., at least two, three, four, or five) comprises a target-hybridizing sequence selected from Table 78 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 752 to 756 (Table 78). At least one Label Extender probe (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 79 (underlined). At least one Label Extender comprises, consists essentially of, or consists of a sequence selected from SEQ ID NOS: 757 to 770 (Table 79). The test may optionally employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 771 to 773 (Table 80).

Table 78: Capture Extender of Mycobacterium tuberculosis SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF Mycobacterium tuberculosis 752. accectaaaucactttceaTTTTTctcttggaaagaaagt 753. ccacaaectcatcccac'TTTTTctcttggaaagaaagt 754. teaccaeacaecctetcaTTTTTctettggaaagaaagt 755. aettaasccataaaattteaceTTTTTctettggaaagaaagt 756. tcgcccgcacgctcacTTTTTctcttggaaagaaagt 46 PCT/U S2010/040649

Table 79: Label Extender of Mycobacterium tuberculosis SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Mycobacterium tuberculosis 757. csccttsgtausccstcaTTTTTfiaagttaccgtttt 758. eecceectaccc-etcetTTTTTctgagtcaaagcat 759. seccetatctcaetcccaets'TTTTTgaagttaccgtttt 760. sctecctcccstaegagtctsTTTTTctgagicaaagcat 761. ccattetecaatattccccacfTTTTTgaagttaccgtttt 762. sctecatcaeecttecscTTTTTctgagtcaaagcat 763. tcccccacecsecatcTTTTTgaagttaccgtttt 764. tacaacc-cgaaggccgtcaTTTTTctgagtcaaagcat 765. tecttcttctccacctaccetcTTTTTgaagttaccgtttt 766. teecacetaetteecceeTTTTTctgagtcaaagcat 767. ctacgtattac-cecsectecTTT'TTgaagttaccgtttt 768. cceeacaacectcecaccTTTTTctgagtcaaagcat 769. ceaectctttacecccaataattTTTTTgaagttaccgtttt 770. aacaacecsacaaaccacctaTTTTTctgagieaaagcat

Table 80: Blocking Label Extender of Mycobacterium tuberculosis SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Mycobacterium tuberculosis 771. ccccac-caacaagctgatagg 772. ttcgtcgatggtgaaagaggtt 773. aatccgagagaacccggaec WO 2012/002964 [0080] in another embodiment, the target polynucleotide is indicative of a spirochete bacterium, for example, Treponema pallidum., which causes syphilis, a sexually transmitted disease. In one embodiment, the target polynucleotide for detecting spirochete infection is a 16S RNA sequence. At least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target-hybridizing sequence selected from Table 81 (underlined). At least one Capture Extender comprises, consists essentially of, or consists of a sequence selected from SEQ ID NOS: 774 to 779 (Table 81). At least one Label Extender (LE) (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 82. At least one Label Extender may comprise, consist essentially of or consist of a sequence selected from SEQ ID NOS: 780 to 791 (Table 82). The test may optionally employ a Blocking Label (BL), e.g., containing the sequence of SEQ ID NO: 792 (Table 83). 47 PCT/US2010/040649

Table 81: Capture Extender of Syphilis SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF Syphilis 774. ccccttacgtgttaccgcgTTTTT ctcttggaaagaaagt 775. ccaggcttaccagtccgccTTTTTctcttggaaagaaagt 776. cctccgtgattctagaccagcaTTTTTctcttggaaagaaagt 777. tic gccaccg^gttcttcTTTTT ctcttggaaagaaagt 778. cgcacctcagcgtcaatcatTTTTT ctcttggaaagaaagt 779. taatgcgttcgcgtcggTTTTT ctcttggaaagaaagt

Table 82: Label Extender of Syphilis SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Syphilis 780. ggggcttattcgcacgactriTTTgaagttaccgtttt 781. gctgctggcacgtaattagccTTTTTctgagtcaaagcat 782. aataattccgaacaacgctcgTTTTTgaagttaccgtttt 783. tgcatgcccittacgcccTTTTT ctgagtcaaagcat 784. ttgagctcggggatttcacaTTTTTgaagttaccgtttt 785. 786. gtacccagtgcaettcccaagTTTTTctgagtcaaagcat cacttggaattccggtttccTTTTTgaagttaccgtttt 787. caaatatctacagattccacccctaTTTTTctgagtcaaagcat 788. tettcgctccccacaccttTTTTTgaagttaccgtttt 789. tgtgaactaccaeggtatctaatccTTTTTctgagtcaaagcat 790. caacaceiagtgtacateglttactgTTTTTgaagttaccgtttt. 791. cgccaagactcatacccTTTTTctgagtcaaagcat

Table 83: Blocking Label Extender of Syphilis SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Syphilis 792. cggceagaaaccegcc WO 2012/002964 100811 In another aspect of the present invention, the method also provides for detection of fungal target polynucleotide. Non-limiting examples of fungal infections include: aspergillosis (Aspergillus fiimigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, and Aspergillus niger); blastomycosis (.Blastomyces dermatitidis); coccidioidomycosis (Coccidioides species): cryptococcosis (Cryptococcus neoformans, C. gattii); histoplasmosis (Histoplasma capsulatum); mucormycosis or zygomycosis (Mucorales molds); paracoccidioidomycosis (Paracoccidioides hrasiliensis), pneumocystis pneumonia (Pneumocystis jiroveci); sporotrichosis (Sporothrix schenckii); etc. 48 PCT/US2010/040649 WO 2012/002964 [0082] In one embodiment, the present invention provides a method for detecting fungal target polynucleotides from an Aspergillus, for example, Aspergillus fumigalus. A. fumigatus is a common, often lethal and difficult to diagnose cause of pathogenic disease in immunocompromised subjects such as in organ transplant recipients, bone marrow transplant and subjects diagnosed with cancer and AIDS. In one embodiment, the target polynucleotide for detecting aspergillus infection is a 188 RNA or 28S RNA sequence. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 84. At least one Capture Extender probe comprises, consists essentially of, or consists of a sequence selected from SEQ ID NOS: 793 to 798 (Table 84). At least, one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from "fable 85. At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 799 to 810 (Table 85). The test may optionally employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 811 to 813 (Table 86).

Table 84: Capture Extender of Aspergillus fumigatus SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF Aspergillus fumigatus 793. actcgcsgtsagscggaTTTTTctcttggaaagaaagt 794. aagatccaaccgaaceagtTTTTTeicttggaaagaaagt 795. ca iaaaattccccagaaggaTTTTTctct tggaaagaaagt 796. cagctgaalactgacaccccTTTTTctc ttggaaa ga aagt 797. gaaaacatccttagegaatgTTTTTctettggaaagaaagt 798. csaacaaatcatcataaaaacTTTTTctcttggaaagaaagt

Table 85: Labe! Extender of Aspergillus fumigatus SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Aspergillus fumigatus 799. aggttcaactacgagctttttaactsTTTTTgaagttaccgtttt 800. ccaaccagccagacccaTTTTTctgagtcaaagcat 801. acctgclttgaacactctaattttTTTTTgaagitaecgittt 802. catactaaigtattegagcaaagTTTTTetgagicaaageat 803. caatcctaaaaaecaaeaaaaiagaTTTTTgaagttaccgtitt 804. ceactatccctattaatcattacggTTTTT ctgagtcaaagcat 805. aaatccaagaatttcacctclga 111 F1 gaagttaccgtttt 806. ctttcgcaatagttagtcttcagcTTTTTctgagtcaaagcat 807. cctaac tttc gltcc c tgattaatTTTTT gaagttaccgtttt 808. cggtatctgatcgtcttcaatccTTTTTctgagtcaaagcat 809. ggcatagtttatggttaagactacgaTTTTTgaagttaccgtttt 81 ft accgcccgatccctagtcTTTTTctgagtcaaagcat 49 PCT/US2010/040649

Table 86: Blocking Label Extender of Aspergillus fumigatus SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Aspergillus fumigatus 811. cceeacagceagtgaaggc 812. ttcacagtaaaagtcctggttcc 813. accgcacgtcctattctattattc WO 2012/002964 [0083] In another embodiment, the fungal target polynucleotides are from Candida, for example, Candida albicans, an opportunistic yeast which causes oral and genital infections in human, particularly in immuno-compromised subjects (e.g. organ and bone marrow transplantation recipients, cancer and AIDS patients). In one embodiment, the target polynucleotide for detecting Candida infection is a 18S or 28S RNA sequence. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 87 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 814 to 819 (Table 87). At least one Label Extender probe (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 88 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 820 to 831 (Table 88). The test may optionally employ a Blocking Label (BL), e.g., at least one or two BLs, such as the BLs of SEQ ID NOS: 832 to 833 (Table 89).

Table 87: Capture Extender of Candida albicans SEQ ID NO CAPT URE EXTENDER NUCLEOTIDE SEQUENCE OF Candida albicans 814. aaaaauatssaccaaccaaTniTctcttggaaagaaagt 815. acccasaageaaaasctceTTTTT'ctcttggaaagaaagt 816. teettceccataaataactTTTTTctcttggaaagaaagt 817. actgatacccccgaccgtcTTTTTctcttggaaagaaagt 818. aaacgtccttggtaaatgcUtcTTTTTctcttggaaagaaagt 819. gtgccgattgcgtcaataaaaTTTTTctcttggaaagaaagt

Table 88: Label Extender of Candida albicans SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Candida albicans 820. gagctttttaactgcaacaactttaataTTTTTgaagttaccgtttt 821. ccaagcccaaggttcaactacTTTTT ctgagtcaaagcat 822. gagcaaaggcctgctttgaaTTTTTgaagttaccgtttt 823. cctattctattattccatgctaatatattcTTTTTctgagtcaaagcat 824. aaccaacaaaata gaaccaiaacgtTTTTT gaagttaccgtttt R9S cctattaatcattacgatggtcctagaTTTTT ctgagtcaaagcat 50 PCT/US2010/040649 SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Candida albicans 826. aeaatttcacctcteacaacteaatTTTTTgaagttaccgtttt 827. gcagtagttagtcttcagtaaatccaTTTTTctgagtcaaagcat 828, cctaactttcgttcttgattaatgaTTTTT gaagttaccgtttt 829. cggtatctgatcatcttcgatcc'ITTTTctgagtcaaagcat 830. ggcatagtttatggttaagactacgaTTTTTgaagttaccgtttt 831. aaacaacaacc gatccctagtcTTTTT etgagtcaaagcat

Table 89: Blocking Label Extender of Candida albicans SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Candida albicans 832. gctgggtceagtacgeatc 833. ca-CtctaaitilltcaLiagtaiiaagtcc WO 2012/002964 [0084] In another embodiment, the fungal target polynucleotides are from Cryptoeoccus. In particular, the target polynucleotides are from Cryptococcus neofor mans, a medically important species of the Crytococcus best known for causing a severe form of meningiti s and meningo-encephaiitis in subjects diagnosed with HIV-AIDS. The fungus is also known to infect and cause moderate to severe disease in patients undergoing cancer chemotherapy and metabolic immunosuppression etc. in one embodiment the target polynucleotide for detecting Cryptoeoccus infection is an 18S RNA sequence. In such embodiments, at least one Capture Extender (e.g., at least two, three, four, or five) comprises a target-hybridizing sequence selected from Table 90 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 834 to 839 (Table 90). At least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 91 (underlined). At least one Label Extender may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 840 to 851 (Table 91), The test may optionally employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 852 to 854 (Table 92).

Table 90: Capture Extender of Crytococcus neoformans SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF Crytococcus neoformans 834. gcctcgccagacctgaagttTTTTT ctcttggaaagaaagt 835. gcactccgtgaggaggaccTTTTTctcttggaaagaaagt 836. ggttcccctgcacacccagTTTTTctcttggaaagaaagt 837. gcgattgcctgctttgaacacTTTTTctcttggaaagaaagt 838. cggcatcgtttactgttaagactaTTTTT ctcttggaaagaaagt 51 PCT/US2010/040649 WO 2012/002964 839. cgtgggccgatecctagtTTTTTctcttggaaagaaagt

Table 91: Label Extender of Crytococcus neoformans SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF Crytococcus neoformans 840. gaggtaaggtccageaagacagtTTTTTgaagttaccgtttt 841. taaagagcatacaggaccaccagTTTTTctgagtcaaagcat 842. tattattccatgctaatgtattcggTTTTTgaagttaccgtttt 843. aaatagaaccgcacgtcctattcTTTTT ctgagtcaaagcat 844. ceeceatcctagaaaccaacaTTTTTgaagttaccgtttt 845. ccgaccgtccctattaatcattaTTTTTctgagtcaaagcat 846. ccgtcaatctaagaatttcacctctaTTTTTgaagttaccgtttt 847. gctttcgcagttgttggtcttTTTTT ctgagtcaaagcat 848. ccctaaccttcgttcttgatcaTTTTTgaagttaccgtttt 849. caacggtatctaategtttttgateTTTTTctgagtcaaagcat 850. gccgacccagtcaaagattsaTTTTTgaagttaccgtttt 851. caaagactttgatttctcgtaaggtTTTTTctgagtcaaagcat

Table 92: Blocking Label Extender of Crytococcus neoformans SEQ ID NO BLOCKING L ABEL EXTENDER NUCLEOTIDE SEQUENCE OF Crytococcus neoformans 852. tctaattttttcaaggtaaaattcct 853. gcaacggaHUicciicitgccc 854. atgaaaacgtccttggeaaat [0085] The present invention provides methods for detection of target polynucleotides from uni- and/or multicellular parasites or protozoa. Non-limiting examples of parasites or protozoa include: leishmaniasis (Leishmania tropica mexicana, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania chagasi); trypanosomiasis (Trypanosoma brucei gambiense. Trypanosoma braced rhodesiense); toxoplasmosis (Toxoplasma gondii); schistosomiasis (Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Schistosoma mekongi, Schistosoma intercalatum); malaria (Plasmodium virax, Plasmodium falciparium, Plasmodium maiariae and Plasmodium ovals); Amebiasis (.Entamoeba histolytica): Babesiosis (Babesiosis microti); Cryptosporidiosis (Cryptosporidium par-sum); Dieniamoebiasis (Dientamoeba fragilis); Giardiasis (Giardia lamblia); Helminthiasis and Trichomonas {Trichomonas vaginalis).

[0086] The method of the present invention described above may include detection of a housekeeping gene polynucleotide, where the level of the housekeeping gene polynucleotide is used for normalization (e.g., across samples) and/or calculation of the copy number of the PCT/US2010/040649 WO 2012/002964 target polynucleotide. In certain embodiments, the housekeeping gene polynucleotide is glyceraidehyde 3-phosphate dehydrogenase (GAPDH) polynucleotide. In such embodiments, at least one Capture Extender probe (e.g., at least two, three, four, or five) comprises a target hybridizing sequence selected from Table 93 (underlined). At least one Capture Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 855 to 858 (Tables 93). In such embodiments, at least one Label Extender (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence shown in Tables, 1, 2 or 94 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 2 to 7 (Table 1), 8 to 13 (Table 2), or 859 to 870 (Table 94). The test may further employ a Blocking Label (BL), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 871 to 875 (Table 95).

Table 93: Capture Extender of GAPDH SEQ ID NO CAPTURE EXTENDER NUCLEOTIDE SEQUENCE OF GAPDH 855. agtcaataaaggggtcattgatTTTTTctcttggaaagaaagt 856. ccttgacagtaceatsaaatTT rTTctcttggaaagaaagt 857. egccccacttgattttgga’ FT ΓΪ Tctcttggaaagaaagt 858. ............... .....................................................

Table 94: Label Extender of GAPDH SEQ ID NO LABEL EXTENDER NUCLEOTIDE SEQUENCE OF GAPDH 859. 860. cggctgactgtcgaacaggaTTTTT ctgagtcaaa gcat 861. gagcgatgtggctcggcTTTTTgaagttaccgittt 862. tcaccti cccca tgstgtciTTTTTe tgagtca aagcat sO 00 ccaggcgcccaatacaacTTTTTgaagttaccgtttt 864. aettaaaaacagccctgatgaTTTTTetgagtcaaagcat 865. tggaaeatgtaaaccatgtagttgaTTTTTgaagttaccgtttt 866. ttgccatgggtggaatcatatTTTTTctgagtcaa ageat 867. tgacaagcttceegttctcagTTTTTgaagttaccgtttt 868. tggigatgggaittccattgaTTTTTctgagteaaagcat 869, gacgtactcagcgccaacatTTTTTgaagttaccgtttt 870. aagaceccagtggactccac ΓΙI T'f ctgagtcaaagcat

Table 95: Blocking Label Extender of GAPDH SEQ ID NO BLOCKING LABEL EXTENDER NUCLEOTIDE SEQUENCE OF GAPDH 871. caaatccgttgactccgacct 53

Capture Extender and/or Label extender probes illustrated or represented by the Tables provided herein. The kit may further comprise at least one Blocking Label probe specific for the target polynucleotide, or a set of Blocking Label probes illustrated or represented by the Tables provided herein. The kit may further comprise at least one or a series of signal-amplifying probes as described. 2010356288 07 Apr 2017 [0088] In some embodiments, the kit further comprises components for conducting a positive control. For example, the positive control may be a cell lysate derived from HeLa cells, a cell-line derived from cervical cancer cells, or any commercially available ceil line having a particular characteristic of interest.

[0089] In some embodiments, the kit further comprises a probe pair or set as a normalization control, to normalize across samples and to calculate the target polynucleotide copy number. The normalization control probes may comprise at least one Capture Extender probe and at least one Label Extender probe specific for one or more housekeeping gene polynucleotides, such as but not limited to, GAPDH (GAPD). Exemplary pairs or sets of Capture Extender probes and Label Extender probes for GAPDH are disclosed in Tables 1,2, 93, and 94 above. The kit may further comprise Blocking Label Extenders disclosed in Table 95 above.

[0090] In certain embodiments, the kit is configured such that the level of target polynucleotide and level of normalization control are read simultaneously or sequentially from a patient sample in the same reaction. Such may be accomplished by employing different labels, such as alkaline phosphatases having different luminescent substrates, or combinations of different signal generating labels (e.g., alkaline phosphatase and horseradish peroxidase). 54 PCT/US2010/040649 WO 2012/002964

EXAMPLES

Example 1; Comparison of currently available HPV-tests [0091] TABLE 96 shows a comparison of the invention with currently available HPV-tests. TABLE 96:

Company and Product HPV test based on Advantages/Disadvantages Present invention Use of primary probes hybridizing to the target sequence without amplifl cation. Signal is intensified by additional probes which hybridize to the primary probes, creating a multilayer probe. No purification of RNA or DNA required from PAP smears. No need for RT-PCT, Amplifies signals, not target. Access Genetics Standard PCR technology Sensitive. Contamination issues Hologic (3ra wave) Invader® HPV 3 specifically designed oligonucleotides and a fluorescent signal detection technology Poor specificity Qiagen/Diagene Hybrid Capture 2® (HC2) assay Enzyme-linked antibody detection of RNA/DNA hybrid technology Requires purification of RNA or DNA. ' RNA/DNA hybrid must be captured on solid phase. Poor specificity and sensitivity Expensive Ventana’s Inform® HPV In-situ hybridization technology Requires intervention by eye Not sensitive

Example 2: Protocol [0092] The schematic in FIG, 1 illustrates the method of the invention. Briefly, the method involves capturing target polynucleotides from a sample by contacting with a Capture PCT/US2010/040649 WO 2012/002964

Extender and Label Extender under hybridizing conditions (e.g,, about 55°C for 120 minutes in 3x SSC, 10% dextransulfate, 0.2% casein, 10 μg/ntL polyA and 100 pg/mL denatured salmon sperm DNA). Signal amplification is then carried out by sequentially hybridizing a pre~Amplifier, Amplifier Probe and Label Probe at 55°C, 55°C and 50°C respectively for 30 minutes each in 3x SSC, 10% dextran-sulfate, 0.2% casein, 10 pg/rnL polyA and 100 pg/mL denatured salmon sperm DNA and with wash buffer: 20mmol/L Tris-HCL, 400 mmol/L lithium chloride, 1 niL/L Tween 20. The Label Probe may be directly conjugated with alkaline phosphatase, or biotinylated, labeled with fluorescein, or other light emitting dyes.

Example 3: Correlation of Relative Light Units (RLU) with HPV type 16 sequence concentration [0093] Samples containing various concentrations of HPV type 16 ss DNA sequences were incubated with Capture Probe-coated plates together with HPV type 16-specific Capture Extender (CE), Label Extender (LE) and Blocking Label (BL) for 1, 2, 3, 4 h and overnight. Probe sets are identified in Tables 3 to 14. TABLE 97 and Figure 2 below show the correlation of RLU with the concentration of viral DNA. TABLE 97: RLU1 RLU2 AVE AVE-BK std 50amol 957.753 957.403 957.578 957.0605 0.25 5amol 862.611 662.097 762.354 761,8365 141.78 O.Samol 220.362 234.933 227.6475 227.13 10.30 0.05amol 42.555 45.472 44.0135 43.496 2.06 O.OOSamol 5.301 5.373 5.337 4.8195 0.05 Background 0.46 0.575 0.5175 0 0.08 [0094] The results show that the method of the invention using the probe set described for detecting HPV has a sensiti vity of about 3000 copies, which, given the high specifi city of the probe set, is sufficient for accurate HPV detection.

Example 4: Detection of HPV in patient samples at various stages of infection [0095] FIG. 3 shows the detection of HPV target polynucleotides using the method of the invention, HPV target polynucleotides in patient Pap smear samples at various stages of HPV infection, i.e. at the C1N (cervical intraepithelial neoplasia) stage 1, 2, or 3; or at the ASC-US (atypical squamous cells of undetermined significance) stage in high risk and low risk natients were detected using the probe sets identified in Tables 3 to 14. 56 PCT/US2010/040649 WO 2012/002964 [0096] The results in TABLE 98 and FIG, 3 show an increase in levels of HPV target polynucleotides in Pap smears of patients with C1N 3 infection. TABLE 98: ASCUS ASCH CIN1 CIN2 CIN3(1) BK High risk 0.8965 6.3085 15.0365 29,804 168.932 0.8675 Low risk 1.1925 0.9135 0.655 2.724 2.8915 0.8225 [0097] ASC, which are abnormal cells of uncertain significance, and CIN1 usually recover spontaneously. The CIN1 have a few cells showing intraepithelial neoplasia which can be precaneerous. CIN3 lesions represent severe neoplasia, involving the full thickness of the mucosa and this class includes carcinoma in situ, a pre-mvasive cancer, CIN3 lesions are less likely to recover spontaneously and commonly progress to invasive cancer. Background levels (BK) were very close to that of the lowest abnormal cell of uncertain significance [0098] Figure 4 shows the detection sensitivity for HIV target polynucleotides (HIV-A, HIV-B, and HIV-F). HIV target polynucleotides were detected using the probe set identified in Tables 60 to 68 in serially diluted plasma samples, spiked with 25, 50, 100 and 200 copies of the HIV target polynucleotide.

[0099] The results show that this embodiment can detect as low as 25 copies. In addition. Figure 4 shows that the assay is linear for samples that are serially diluted.

Example 6: Detection Sensitivity tor IIB V

[00100] Figure 5 shows the sensitivity of detection for HBV target polynucleotides. HBV target polynucleotides were detected using the probe set from Table 57, Table 58 and Table 59.

[00101] The results show that the method of detection in this embodiment, measured by log relative light units (RLU), is linearly correlated with the log number of copies of HBV. 57 PCT/U S2010/040649 WO 2012/002964

Example 7: Detection Sensitivity for HCV

[00102] Figures 6A and B show the sensitivity of detection for HCV target polynucleotides. HCV target polynucleotides were detected using the probe set from Table 72, Table 73 and Table 74.

[00103] Figure 6A shows that, in this embodiment, log relative light units (RLU) is linearly correlated with the log number of copies of HCV.

[00104] Figui •e 6B shows the quantitation of HCV in 6 patient samples.

Example 8: Detection Sensitivity for Coronavirus (SARS) [00105] Figure 7 shows the detection sensitivity for coronavirus (SARS) target polynucleotides, SARS target polynucleotides were detected using the probe sets from Table 75, Table 76 and Table 77.

Example: 9 Lack of Cross-Reactivity for H1NI Assay [00106] An assay for hemaglutinin-neuraminidase (H1N1) of influenza virus was conducted. The H1N1 target polynucleotides were detected using the following probe sets from Table 69, Table 70 and Table 71.

[00107] 'FABLE 99 shows that the H1N1 assay does not cross-react with various strains of Influenza A, influenza B or other respiratory' virus. TABLE 99:

Viruses Tested in Samples Result Influenza A; Brisbane/10/2007 H3N2 negative Brishane/59/2007 H1N1 negative Hong Kong/29/2006 negative Netherlands/!34/04 HI negative New' Caledonia/20/99 H1N1 negative Solomon Isiands/03/2006 H1N1 negative Taiwan/42/2006 negative Victoria/3/75 H3 negative Wisconsin/67/05/H3N2 negative Si.ngapore/63/04 negative influenza B: Florida/04/2006 negative 58 PCT/US2010/040649

Viruses Tested in Samples Result Florida/07/2004 negative Malaysia/25 06/2004 negative Panama/45/90 negative Yamanashi/166/98 negative Other Respiratory Viruses: Parainfluenza 1 negative Parainfluenza 2 negative Parainfluenza 3 negative Parainfluenza 4a negative Parainfluenza 4b negative Corona virus 229E negative Coronavirus NL63 negative Coron a virus OC43 negative SARS (200300592) negative WO 2012/002964

Example: 10 ssDNA complementary to 18S RNA or Fungal cells as standard materials [00108] Figure 8 shows the correlation between the numbers of fungal cells and 18S RNA copies using the probe sets described in Tables 84 to 89. Both fungal cells and ssDNA complementary to 18S RNA were tested for their usefulness as standard materials. The results show a correlation between the numbers of fungal cells and 18S RNA copies with the level of luminescence (R> 0.95) detected.

Example: 11 Quantifying different species and strains of Aspergillus and Candida [00109] Figure 9 shows the quantitation of different species and strains of Aspergillus and Candida. Various species of Aspergillus and Candida were detected using the 18S RNA probe set for Aspergillus (Table 84, Table 85, Table 86) and for Candida (Table 87, Table 88, and Table 89 respectively and the method described above.

[00110] The results show that the method of detection measured by log relative light units (RLU) can detect as few as 10 cells/well and there is no cross-reactivity between the 16S RNA probes from Aspergillus and Candida. The results show7 that the Aspergillus 16S RNA probe-set consistently detects various species of Aspergillus and not Candida, and the Candida 16S RNA probe-set detects only the Candida species and not Aspergillus. On the other hand, the common 18S RNA probe-set can detect either Candida or Aspergillus of various strains allowing for a single test that detects many potential fungal pathogens. 59 PCT/US2010/040649 WO 2012/002964

Example: 12 Quantification Candida in Clinical Blood Samples [00111] Figure 10 shows the quantification of Candida, Clinical blood samples from patients infected with Candida were assayed for the Candida target polynucleotides using the IBS RNA probe set Candida and a “common probe” from Table 84. Table 85, Table 86, Table 87, Table 88, and Table 89.

[00112] The results show detection measured by log relative light units (R.LU). A 50 ul blood sample for an uninfected patient (negative) or infected patient blood sample (positive) was tested. The clinical culture within 24-48 hr showed that two of them were negative and one positive. The results (Fig 10) of QuantiFungiIjVi assay, which took only 5 hours, were consisten t with the results of clinical microbes culture.

[0100] All publications, patents and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0101] The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will he obvious to those skilled in the art. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[0102] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary' skill in the art to which this invention belongs.

[0103] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. 60

Claims (12)

1. We claim:

   1. A method for determining the presence, absence, or level of HPV type-16 in a sample, consisting of the following steps: a. capturing a target polynucleotide, if present, from a sample by hybridisation to a set of capture extender oligonucleotides probes consisting of SEQ ID NOs: 14 to 21 and wherein the target polynucleotide is indicative of the presence of said HPV type 16; b. hybridising said target polynucleotide with a set of blocking oligonucleotides probes consisting of SEQ ID NOs: 38 to 48; and c. detecting the target polynucleotide by hybridisation with a set of label extender oligonucleotides probes consisting of SEQ ID NOs: 22 to 37; and wherein said probes in steps (a), (b) and (c) for detecting HPV have a sensitivity of about 3000 copies.
2. 2. The method of claim 1, wherein said sample is a biological sample.
3. 3. The method of claim 2, wherein said biological sample is a pap smear.
4. 4. The method of claim 1, wherein said capturing is done by hybridising said sample with said set of capture extender oligonucleotides and a capture probe that hybridises to said set of capture extender oligonucleotides and is attached to a solid support.
5. 5. The method of claim 4, wherein said solid support is a bead.
6. 6. The method of claim 4, wherein said solid support is a well of a plate.
7. 7. The method of claim 1, wherein said detecting is done by hybridising said set of extender oligonucleotides with an oligonucleotide probe that is directly or indirectly labeled.
8. 8. The method of claim 7, wherein said oligonucleotide probe is directly or indirectly labeled with a label that is luminescent.
9. 9. The method of claim 7, wherein said oligonucleotide probe is directly or indirectly labeled with a label that is chromogenic.
10. 10. The method of claim 7, wherein said oligonucleotide probe is directly or indirectly labeled with a label that is fluorescent.
11. 11. The method of claim 1, wherein, said capturing is done in the presence of oligonucleotides defined by SEQ ID NOS: 49-56; said hybridising is done in the presence of oligonucleotides defined by SEQ ID NOS: 57-72; and said detecting is done in the presence of oligonucleotides defined by SEQ ID NOS: 73-75.
12. 12. The method of claim 1, wherein, said capturing is done in the presence of further oligonucleotides that specifically detect HPV strains 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.