Patent Application
20120003625
This invention relates to detecting the presence, absence, or level of an infectious agent in a sample.
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 case 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.
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 38:104-108 (2009); Ginocchio et al., Comparison of the Third Wave Invader Human Papilloma (HPV) Assay and the Digene HPV 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.
Other tests for HPV include GenProbe's Aptima HPV test, Third Wave's Invader HPV DNA test, Ventana's Inform1 HPV in-situ hybridization test, and other PCR-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 acids 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 over or cross contamination between samples.
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.
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.
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.
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).
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. Consecutive 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., relative to the number of hybridized Label Extender probes).
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.
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 glyceraldehyde 3-phosphate dehydrogenase (GAPDH/GAPD).
Other aspects and embodiments of the invention will be apparent to the skilled artisan in view of the following detailed description.
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, 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.
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.
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.
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 DNA, e.g., may include a pre-Amplifier probe, an Amplifier probe, and a Label probe.
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 such as toilet seats, pond water, tree sap, and insect or plant extracts. Other types of samples 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.
The method of detection may be carried out using the principles set forth in U.S. Pat. No. 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.
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).
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 avoid significant structural constraints. The linking sequence may be short, for example, from about 2 to about 10 nucleotides. The linking sequence may be poly(T) or poly(A) in certain embodiments.
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.
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.
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 poly(T) or poly(A) in certain embodiments.
Various Label Extender sequences (including Label Extender probe sets) for the detection of certain infectious agents are disclosed herein. See Tables 1, 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 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.
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 3×SSC, or comparable hybridization buffer.
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 agents 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.
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-Amplifier 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 3×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′ AGGCATAGGACCCGTGTCTttttttttttAGGCATAGGACCCGTGTCTtttttATGCTTTGACTCA GAAAACGGTAACTTC 3′ (SEQ ID NO:1). The underlined sequences are complementary to sequences in the GAPD Label Extenders (used as a positive control as described herein).
The configuration and hybridization of test components, such as Label Extenders, may be as described in U.S. Pat. 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
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 may be used as a control for normalizing across samples, and for quantifying polynucleotide copy number.
The sequences complementary to the preamplifier sequence of SEQ ID NO: 1 is underlined for each of the Label Extenders shown in Table 1.
ctgagtcaaagcatTTTTTttctccatggtggtgaagacg
ctgagtcaaagcatTTTTTgcaggaggcattgctgatga
ctgagtcaaagcatTTTTTcacagccttggcagcgc
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.
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
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.
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 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 Label 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.
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.
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.
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.
In various embodiments, the target polynucleotide is indicative of the presence of a virus 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 mild or acute respiratory diseases); Parvoviridae; Rhabdoviridae (e.g., rabies); Filoviridae (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; Picornaviridae; Caliciviridae (e.g., winter vomiting disease); Astroviridae (e.g., gastroenteritis); Retroviridae (e.g., HIV, HTLV) and Reoviridae (e.g., Colorado Tick fever).
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.
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.
In various embodiments, the target polynucleotide sequence is a portion of an HPV late viral gene (e.g., L1 and/or L2), or an early viral gene (e.g., E1 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.
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 five) comprise a target-hybridizing sequence selected from Table 3 (underlined). At 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 (BL), 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).
ctcctgtgggtcctgaaacattTTTTTctcttggaaagaaagt
cacgtcgcagtaactgttgcttTTTTTctcttggaaagaaagt
tgttgttccatacaaactataacaataatTTTTTctcttggaaa
aatctaacatatattcatgcaatgtaggTTTTTctcttggaaag
atattgtaatgggctctgtccgTTTTTctcttggaaagaaagt
cccattaacaggtcttccaaagtaTTTTTctcttggaaagaaa
ggtagattatggtttctgagaacagatTTTTTctcttggaaaga
cccgtaccctcttccccattTTTTTctcttggaaggaaagt
gtctgcttttatactaaccggtttcTTTTTgaagttaccgtttt
gcagttctcttttggtgcataaaatTTTTTctgagtcaaagcat
aactgtggtaactttctgggtcgTTTTTgaagttaccgtttt
agttgtttgcagctctgtgcatTTTTTctgagtcaaagcat
ttcccatctctatatactatgcataaatTTTTTgaagttaccgt
aacatttatcacatacagcatatggaTTTTTctgagtcaaagc
cggtttgttgtattgctgttctaaTTTTTgaagttaccgtttt
taatacacctaattaacaaatcacacaaTTTTTctgagtcaaag
ggacacagtggcttttgacagtTTTTTgaagttaccgtttt
ccagatgtctttgcttttcttcaTTTTTctgagtcaaagcat
gatctgcaacaagacatacatcgaTTTTTgaagttaccgtttt
tgggtttctctacgtgttcttgatTTTTTctgagtcaaagcat
gagatcagttgtctctggttgcaTTTTTgaagttaccgtttt
gctgtcatttaattgctcataacagtaTTTTTctgagtcaaagc
tcacacttgcaacaaaaggttacaTTTTTgaagttaccgtttt
cgcacaaccgaagcgtagagTTTTTctgagtcaaagcat
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).
cgtttttcattaaggtgtctaagtttttTTTTTctcttggaaag
tgctcggttgcagcacgTTTTTctcttggaaagaaagt
tgttgccttaggtccatgcataTTTTTctcttggaaagaaagt
gctttctactactagcttaattctggcTTTTTctcttggaaaga
ctggatcagccattgttgcttTTTTTctcttggaaagaaagt
tactacctgctaatcttctatgagcttTTTTTctcttggaaaga
tggccagggaagatccactTTTTTctcttggaaagaaagt
ttgcagctcaatacaaaacggTTTTTctcttggaaagaaagt
cccagctatgttgtggaatcgtTTTTTgaagttaccgtttt
aatggcactggcctctatagtgTTTTTctgagtcaaagcat
tcgttggagtctttcctgtcgTTTTTgaagttaccgtttt
cttaatattatacttgtgtttctctgcgTTTTTctgagtcaaag
taaatgcaatacaatgtcttgcaaTTTTTgaagttaccgtttt
ggaatttcattttggggctcTTTTTctgagtcaaagcat
gctcgtgacatagaaggtcaaccTTTTTgaagttaccgtttt
tttcttcctctgagtcgcttaattTTTTTctgagtcaaagcat
atgattaactccatctatttcatcgtTTTTTgaagttaccgtt
cgtcgggctggtaaatgttgTTTTTctgagtcaaagcat
gctcgaaggtcgtctgctgaTTTTTgaagttaccgtttt
tgttcagaaacagctgctggaatTTTTTctgagtcaaagcat
cggacacacaaaggacagggTTTTTgaagttaccgtttt
actgctgggatgcacaccaTTTTTctgagtcaaagcat
cgtcagtttcctcatctgaaaacttTTTTTgaagttaccgtttt
cgtcaagtcatttaagcttggtaccTTTTTctgagtcaaagcat
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).
gtctttctgcaggatttttgaacTTTTTctcttggaaagaaagt
gcttagttcatgcaatttccgagTTTTTctcttggaaagaaagt
cctctgtttctgttaactgacctttgTTTTTctcttggaaagaa
atctaaattcacttacttttgaataaaatTTTTTctcttggaaa
catatacctttgtttgtcaatttttctTTTTTctcttggaaaga
acgcatgtttacacttgggtttTTTTTctcttggaaagaaagt
tctgagctgtcgggtaattgcTTTTTctcttggaaagaaagt
gtagggtatttccaatgccgaTTTTTgaagttaccgtttt
cagtagacacaattcaatcttagttcatcTTTTTctgagtcaaa
gtggtgtgtcgtccctatatactattTTTTTgaagttaccgtt
cttaaacattttgtacacactccgtTTTTTctgagtcaaagcat
acacgttatacacctaattaacaaatcaTTTTTgaagttaccgt
tcttctggacacaacggtctttgTTTTTctgagtcaaagcat
tctttttatccaaatgtctttgttttTTTTTgaagttaccgtt
ttcctcctatgttgtggaatcgttTTTTTctgagtcaaagcat
cttgcaacgtaggtgtttctccTTTTTgaagttaccgtttt
ctcaggttgcaaatctaacacatagtTTTTTctgagtcaaagc
ggactgtctatgacatcctcctcaTTTTTgaagttaccgtttt
ccggttctgcttgtccagctTTTTTctgagtcaaagcat
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 probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 106 to 117 (Table 13). 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: 118 to 127 (Table 14).
caaacaaatttcatggatgctttcTTTTTctcttggaaagaaa
tactccatatggctggccttcTTTTTctcttggaaagaaagt
tgtttttctaacgtttctccatacacTTTTTctcttggaaagaa
caccgtccaccgatgttatgTTTTTctcttggaaagaaagt
tccagctggaccgtcaatagtatTTTTTctcttggaaagaaagt
ccattaataaatcttccaatttacgtaTTTTTctcttggaaaga
gcagtttgtaaggtcgttcagctTTTTTgaagttaccgtttt
ttctacctcgttgcacaaatcatTTTTTctgagtcaaagcat
taattcttgtttgcagtatacacaattTTTTTgaagttaccgtt
aaagtcatatacctcactccgctgTTTTTctgagtcaaagcat
aataaatgacataactgtttgttgcatTTTTTgaagttaccgtt
ggtttttgacatgtaatacacctaattTTTTTctgagtcaaagc
tctaaaacatagtcttgcaatgtagttatTTTTTgaagttaccg
agttgcctcgggttccaaaTTTTTctgagtcaaagcat
acacaattgctcataacagtataggtcTTTTTgaagttaccgtt
cttcctcctcctctgagctgtcTTTTTctgagtcaaagcat
ggaggtgtctggttttgcttgTTTTTgaagttaccgtttt
ttacaacaggacgttacaatattataattTTTTTctgagtcaaa
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).
tacctcggtttgctgtagtggTTTTTctcttggaaagaaagt
ggttccccgtccctatatactaTTTTTctcttggaaagaaagt
tttatgaaatcttcgtttgctatttTTTTTctcttggaaagaaa
ggtccacgcatatctgatgttataTTTTTctcttggaaagaaa
tttcctgcaaggtgggctttTTTTTctcttggaaagaaagt
ccgtgaggcttctactaccagctTTTTTctcttggaaagaaagt
acaaatcctagtgagtccataaacagTTTTTctcttggaaagaa
cccgtattttagcataaaattttataTTTTTctgagtcaaagc
ccgagtccgagtaatatcgtagctTTTTTgaagttaccgtttt
ttagttatattttctaatgtagttgcatacaTTTTTctgagtca
cacagcggtttcagacaacacaTTTTTgaagttaccgtttt
aggtgtcttaatttttctgctggaTTTTTctgagtcaaagcat
ttcattgtaaggacataaatctaatacaaTTTTTgaagttaccg
catacaaggtcaaccggctgtatTTTTTctgagtcaaagcat
gtcgggttcatctatttcatcctTTTTTgaagttaccgtttt
ttgatgttggtgattaactgcatgTTTTTctgagtcaaagcat
ggttcatcccgtctggctagtagTTTTTgaagttaccgtttt
gaacactgtattgtgtgacgctgtTTTTTctgagtcaaagcat
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 (e.g., 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).
tatacctctgtgcgttccaatgtTTTTTctcttggaaagaaagt
tataaataaatctttaaaagcaaattgaTTTTTctcttggaaag
cagtgttgctcggggtccaTTTTTctcttggaaagaaagt
tgactaacgccatctgcttcatTTTTTctcttggaaagaaagt
agctcaattctgccgtcacacTTTTTctcttggaaagaaagt
catctgccgagctctctactgtaTTTTTctcttggaaagaaagt
tttctgctgggttcaatgattTTTTTgaagttaccgtttt
cgtttgtccttaaggtgtctacgttTTTTTctgagtcaaagcat
tgtattacactgccctcggtactgTTTTTgaagttaccgtttt
gccgtgcctggtcacaacaTTTTTctgagtcaaagcat
cctacgtctgcgaagtctttcttTTTTTgaagttaccgtttt
tgcatacttattgctatacttgtgtttcTTTTTctgagtcaaag
ttccaaatgcaatacaatttcttgTTTTTgaagttaccgtttt
caacaggatctaattcattctgaggTTTTTctgagtcaaagcat
ttaattgctcgtaacacaacaggtTTTTTgaagttaccgtttt
cgttttcctcctctgactcgcTTTTTctgagtcaaagcat
tcgggctggtagttgtgcaTTTTTgaagttaccgtttt
cgctgtggttcggctcgTTTTTctgagtcaaagcat
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).
ggtctttccctcttgtcttcgaaTTTTTctcttggaaagaaagt
tgcatagaaacgttcaaagcttcTTTTTctcttggaaagaaagt
tttgaataaaacagtaaacattgtttgTTTTTctcttggaaaga
tcgataaatcatataagctttttttagtaaTTTTTctcttggaa
cgcattgccccgtccaaTTTTTctcttggaaagaaagt
cgtgtacgttgccagcaattagTTTTTctcttggaaagaaagt
ggagcttcaattctgtaacacgtaTTTTTctcttggaaagaaa
ttacaatacacacacactacctgtatattgTTTTTgaagttaccg
ttatatacatctgctctacataattcctttTTTTTctgagtcaaa
atacaatcttaatttcagtaaatgctacaTTTTTgaagttaccgt
catactgcatatggattattatccctatTTTTTctgagtcaaagc
acctgctataacgtctatactctctaattTTTTTgaagttaccgt
ttgcctctaatgtagtaccatacacagTTTTTctgagtcaaagc
gtggtctttgacatctatgacaccttaTTTTTgaagttaccgtt
tttgcttttcttcaggcccaaTTTTTctgagtcaaagcat
cacttgggtttcgttacgttgtTTTTTgaagttaccgtttt
cattaccacgcatggctttattaTTTTTctgagtcaaagcat
tgcaatactacatcttttaattgtggtaTTTTTgaagttaccgtt
agtcaatttcagtctgtggttgttaaaTTTTTctgagtcaaagc
tcaaattgctcgtagcattgcaTTTTTgaagttaccgtttt
tacttcatcctcctcctctgagctgTTTTTctgagtcaaagcat
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 SEQ ID NOS: 207 to 212 (Tables 24). 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 25 (underlined). At least one Label Extender probe may comprise, consist essentially of, or consist of a sequence selected from SEQ ID NOS: 213 to 224 (Table 25). 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: 225 to 238 (Table 26).
gtgcagggtccggggtcTTTTTctcttggaaagaaagt
cactctgaacagcgccctgtTTTTTctcttggaaagaaagt
cacaggtcggggtctccaaTTTTTctcttggaaagaaagt
cagtgctatgaatgcatagccgTTTTTctcttggaaagaaagt
tgtgcccaacagcatttgcTTTTTctcttggaaagaaagt
ttcagggtcctccattgcagTTTTTctcttggaaagaaagt
cttccagcacctcacacaattcTTTTTgaagttaccgtttt
gccttatttcatgcaccgattTTTTTctgagtcaaagcat
tttgcactgcacacactgcaTTTTTgaagttaccgtttt
tatacctctcttcgttgtagctctttTTTTTctgagtcaaagc
tttctttttcttcaggacataatggTTTTTgaagttaccgtttt
tcgcttgtttgcattaacatgtcTTTTTctgagtcaaagcat
cgcatgacgttacacttgggtTTTTTgaagttaccgtttt
aatcttttatagttgctttgtctccaTTTTTctgagtcaaagc
gccggtccacaccatctgtatTTTTTgaagttaccgtttt
gcttgttctgcttgtccatctgTTTTTctgagtcaaagcat
atgtcacaatgtagtaattgcttgtgTTTTTgaagttaccgtt
tagtgtgctatcacaactgtgacaatTTTTTctgagtcaaagc
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).
gttagttcttttttgcaatatacacatgTTTTTctcttggaaag
atgcaaaattatatacctcagcacgtTTTTTctcttggaaagaa
gcacactgcataaggaaaatcaTTTTTctcttggaaagaaagt
cacaatgcaattgcttttcctcTTTTTctcttggaaagaaagt
ctattagatgaaatcgtctttttctgtTTTTTctcttggaaaga
gtctccagcaccccaaacatTTTTTctcttggaaagaaagt
aggtcaatttctgtttgaggtgttaTTTTTctcttggaaagaaa
catacactgaatagtcataatacctatatttTTTTTgaagttac
gttttttagttatactttctagtgtagctcTTTTTctgagtcaa
gtagcaccttattaataaatcacataactTTTTTgaagttaccg
cggagttaacggactttgacatctTTTTTctgagtcaaagcat
tgtagattctctaggttctctagatgtttTTTTTgaagttaccg
ttggtactttaccatgcatgattatacTTTTTctgagtcaaagc
cctcatcctcatcctctgagctTTTTTgaagttaccgtttt
gctcctgcaaatggtctacttcatTTTTTctgagtcaaagcat
cttgtctagcttgctgtggccTTTTTgaagttaccgtttt
gtgtattaggtaacacgtatgttgtttagTTTTTctgagtcaaa
cacaaacttacactcacaacaaggtacTTTTTgaagttaccgtt
ggtactgtgaatgtccaactgcacTTTTTctgagtcaaagcat
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 (Table 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).
ctcctctgcgtcctggaacaTTTTTctcttggaaagaaagt
tcaattcgatttcatgcacagaTTTTTctcttggaaagaaagt
gtctttttgcattcaacgcattTTTTTctcttggaaagaaagt
cactattcttaaatctgcaaatacaaagTTTTTctcttggaaag
agcaatcgtaagcacactttacatacTTTTTctcttggaaagaa
attaatatttcatttaaacacttttttagtgtTTTTTctcttgg
ccgtccaagcctatttcatcctTTTTTctcttggaaagaaagt
atcatgcaatgtccgtggtttTTTTTgaagttaccgtttt
tgtctccaacgcctgacacaaTTTTTctgagtcaaagcat
ataattataatgtctatactcacttattttagatTTTTTgaagt
ttgttctaatgtgtctccatatagcgaTTTTTctgagtcaaagc
caatggtctttgacaaataatacatctaTTTTTgaagttaccgt
tgcctttttttttcttgtggacaTTTTTctgagtcaaagcat
cctgtccaacgacccgaaatatTTTTTgaagttaccgtttt
tccaacacactgcacagcgcTTTTTctgagtcaaagcat
tgtgtttgtctacgtcggggtcTTTTTgaagttaccgtttt
atggcgttgttacaggttacactTTTTTctgagtcaaagcat
tcatagcagaataggtcagttggtTTTTTgaagttaccgtttt
cgtctgagctgtcacataattgcTTTTTctgagtcaaagcat
In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from HPV type 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). 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 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).
ggatcctcaaagcgtgccaTTTTTctcttggaaagaaagt
tgatacgaatatcatgcagaggTTTTTctcttggaaagaaagt
tctataacagcgtatcagcagctcTTTTTctcttggaaagaaa
tgttggacatagaggttttaggcaTTTTTctcttggaaagaaa
taggtgtcttgctcgggtccTTTTTctcttggaaagaaagt
aaagtgttgcttttggtccatgTTTTTctcttggaaagaaagt
cccctttgcaaaacacacaatTTTTTgaagttaccgtttt
tcaaatacctctctttcttgcagttTTTTTctgagtcaaagcat
cctctaatgtttctccatacacggTTTTTgaagttaccgtttt
atgtaacggtgtcttggtttcagTTTTTctgagtcaaagcat
gcgcttgtcgttgctgtctTTTTTgaagttaccgtttt
cattgttttacaccagtgtttcactacTTTTTctgagtcaaagc
ggttccaaatctaaaacaatgtcacTTTTTgaagttaccgtttt
aaggtcaacttcctcataattttgtTTTTTctgagtcaaagcat
tcaggtaattgctcgtagcacacTTTTTgaagttaccgtttt
ttttcattctcggagtcggagTTTTTctgagtcaaagcat
gcgaggtttctactactagctgaagTTTTTgaagttaccgtttt
aaggctcgcaatccgtcttTTTTTctgagtcaaagcat
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., at least 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).
gtcatgcaatgtagtgtccaatgtTTTTTctcttggaaagaaagt
tgttaggtgccttagtattctgcTTTTTctcttggaaagaaagt
cgcttactggtccagcagtgcTTTTTctcttggaaagaaagt
cggtgggctttggtccatgTTTTTctcttggaaagaaagt
atagctctaacacaatttcctgcaTTTTTctcttggaaagaaagt
cccgcgacgcttctactactTTTTTctcttggaaagaaagt
caaaatttagtgagtccataaacagcTTTTTctcttggaaagaaagt
cttctgcaatagacacagtctattgtaacTTTTTgaagttaccgtttt
catatacctctgtccgttgtagttgcTTTTTctgagtcaaagcat
atttaatacatgattggcatgcagTTTTTgaagttaccgtttt
cgtagttcccgtattttagcataaaTTTTTctgagtcaaagcat
gcatacaccgattccgagtaatatTTTTTgaagttaccgtttt
ctttgtattagttatggtttctaatgtagttTTTTTctgagtcaaagcat
actcatgcaccttatcaataaattatataaTTTTTgaagttaccgtttt
tggacacaatggtttcaggcaTTTTTctgagtcaaagcat
cggctgtatttcattgtatggacTTTTTgaagttaccgtttt
tgctcgtgacatacaaggtcaacTTTTTctgagtcaaagcat
ctatttcatcgtctgaatctcctaatTTTTTgaagttaccgtttt
aactgcatggtcgggttcatTTTTTctgagtcaaagcat
gctgttgttcgtcccgtctgTTTTTgaagttaccgtttt
caacacagacactgaattctgtgacTTTTTctgagtcaaagcat
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).
tggatagccgcctcgaaaTTTTTctcttggaaagaaagt
cacgcgcaggctgcataTTTTTctcttggaaagaaagt
tttttccatgaaattctaggcagTTTTTctcttggaaagaaagt
taggcagcgacccttccaTTTTTctcttggaaagaaagt
caatatcctttagggtaacatgtcttcTTTTTctcttggaaagaaagt
cagaagctgttgcacttctctgaTTTTTctcttggaaagaaagt
ggtcttcggtgcgcagatgTTTTTctcttggaaagaaagt
attaatttgcaacgtatgcatagataTTTTTgaagttaccgtttt
gtgcattcttgcaaaacacacaTTTTTctgagtcaaagcat
tatgaataaatctctgctgtggtcaTTTTTgaagttaccgtttt
caggacctttaggtgtttatatgcaTTTTTctgagtcaaagcat
tcttcaactgttgttgcatatccaTTTTTgaagttaccgtttt
cacgtctaagatgtcttgtttagtttctTTTTTctgagtcaaagcat
cgccttggttagtatatgttttacctTTTTTgaagttaccgtttt
cgtacaatttagctttatgaaccgTTTTTctgagtcaaagcat
ggtctggaggttgcaggtctaataTTTTTgaagttaccgttttt
tgctcatagcaatgtaaccctacagTTTTTctgagtcaaagcat
acctcatcttctgagctgtctactaatTTTTTgaagttaccgtttt
cttgtccgtccacttcgtccTTTTTctgagtcaaagcat
cagtcgaacgttgctgtcacatTTTTTgaagttaccgtttt
tgtctgtttctgtacactgcacaacTTTTTctgagtcaaagcat
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).
caaagaaagattaaacgtcttgcacTTTTTctcttggaaagaaagt
cgcactgaatttgcagagtgtgTTTTTctcttggaaagaaagt
tggttttctcttctactgtaggtgcTTTTTctcttggaaagaaagt
cgaattaacacttttaaaatatcttcatTTTTTctcttggaaagaaagt
agcgtgcctttcccaatatgTTTTTctcttggaaagaaagt
accctacagggtcaggaggctTTTTTctcttggaaagaaagt
gtgcgtcttgtttgtccacctTTTTTctcttggaaagaaagt
tggaggcatctttactttccataaTTTTTgaagttaccgtttt
aactggtctatagatgttgcagacgTTTTTctgagtcaaagcat
atatgcatatatctctgcggtggTTTTTgaagttaccgtttt
acacaacctttaggttcttataggcTTTTTctgagtcaaagcat
aagcaacaggcacacgctgTTTTTgaagttaccgtttt
ggttaattttcccttgcagttctTTTTTctgagtcaaagcat
cggcttgtgacacaggtaacaaTTTTTgaagttaccgtttt
tgctttagtttttctatttcacacaaTTTTTctgagtcaaagcat
cttccactggttatttagttttatgaTTTTTgaagttaccgtttt
ccagcagtgtaagcaacgaccTTTTTctgagtcaaagcat
gtcttctaattgctcatagcaatgtaTTTTTgaagttaccgtttt
tgtccacctcatcttctgagctTTTTTctgagtcaaagcat
gtatttggtaatgttgtgttaaaggttTTTTTgaagttaccgtttt
cacatccacagcaacaggtcaTTTTTctgagtcaaagcat
caaccagtcggacgttgctgtTTTTTgaagttaccgtttt
atgtctccgtctgtgcactccaTTTTTctgagtcaaagcat
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., 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).
tcatacagggtcctggcctgTTTTTctcttggaaagaaagt
ggaccgtcttgcaaaacacacTTTTTctcttggaaagaaagt
cgtggacatgcggcgtgtTTTTTctcttggaaagaaagt
aaagaattgcgtcttctttacaatatTTTTTctcttggaaagaaagt
agtttagacatacaggttcagggtgTTTTTctcttggaaagaaagt
acaccgagttactactttaaatgattgTTTTTctcttggaaagaaagt
tgatggaacaatgcactgctaagTTTTTctcttggaaagaaagt
tgtaatattgcactggtcacacagtTTTTTgaagttaccgtttt
aatcaatttgcaacgtaggcaaTTTTTctgagtcaaagcat
ggccagtacctcagctgtttttaTTTTTgaagttaccgtttt
cacaacatataactctctaaaggcaaaTTTTTctgagtcaaagcat
ccgtgcaggtccaggcacTTTTTgaagttaccgtttt
atctaaagtttctgtattggtttacttttTTTTTctgagtcaaagcat
gttaatcctgtctcttcttccacgTTTTTgaagttaccgtttt
cagcatctaatccttacttgtaaaatgTTTTTctgagtcaaagcat
ccctgtccacgaatcttttaatttTTTTTgaagttaccgtttt
catttcttccagcaatgtagacagtaTTTTTctgagtcaaagcat
gagctgtctaattgctcgttgcTTTTTgaagttaccgtttt
ctgttcatggtcatcttctgagtctTTTTTctgagtcaaagcat
tctactgtgtaagctgtctagttggtcTTTTTgaagttaccgtttt
cgtgggttgctcacgctcTTTTTctgagtcaaagcat
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 Table 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).
acattctgccttaactgggaccTTTTTctcttggaaagaaagt
cctgttaagtgctttttgcaccTTTTTctcttggaaagaaagt
acgcgagcacctctgcgTTTTTctcttggaaagaaagt
caatataaattgaaatcttgtacctgtatcTTTTTctcttggaaagaaagt
cattgtcctctgcaatgcgtaTTTTTctcttggaaagaaagt
cgctgtatgtcctgtttggctTTTTTctcttggaaagaaagt
cttatgtccgcctctgtacactgTTTTTctcttggaaagaaagt
ctgtgatgaggcagatgtacctgTTTTTgaagttaccgtttt
tacacaattggtataatgtgcgtggTTTTTctgagtcaaagcat
gcaatgtcagcccaaattcctTTTTTgaagttaccgtttt
aaatgcaggaaatctgtaaattccTTTTTctgagtcaaagcat
caaaatgctgatctttcgtagtgtTTTTTgaagttaccgtttt
agtccagtttctttctccactgtatacTTTTTctgagtcaaagcat
gataacggcttttgacacaaggTTTTTgaagttaccgtttt
aatatgatggtttttttcgctctgtTTTTTctgagtcaaagcat
atgtcaaacaaaacaatgtcctttaTTTTTgaagttaccgtttt
atgggtgtctcacacgttggtTTTTTctgagtcaaagcat
caaaagcatctgttgcaggtttTTTTTgaagttaccgtttt
ggacacacaatatccagtgtgccTTTTTctgagtcaaagcat
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 Table 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), e.g., at least one, two, or three BLs each containing a sequence selected from SEQ ID NOS: 483 to 488 (Table 53).
agcatgcagcaaacggatatcTTTTTctcttggaaagaaagt
ccatgaaactgtagacaggccaTTTTTctcttggaaagaaagt
tcaaagtgcctatattgacttatttttTTTTTctcttggaaagaaagt
acagtatctgcatatgctgcgtagTTTTTctcttggaaagaaagt
atgcatgatttccagcaatgtagTTTTTctcttggaaagaaagt
ttaatatacccaacagcaggTTTTTctcttggaaagaaagt
catcacacaactcaaatatagtccgTTTTTgaagttaccgtttt
gcagagtaggcaaagttatgttacactTTTTTctgagtcaaagcat
acttccgtggtaagtaaccacttcTTTTTgaagttaccgtttt
tttaaatctctaaatgcaaacgataatTTTTTctgagtcaaagcat
aacactgtttgcttagtttcttcttctTTTTTgaagttaccgtttt
cttacagcatctaatgcacaaatcaTTTTTctgagtcaaagcat
tggtgataatggcttgtggcaTTTTTgaagttaccgtttt
gcacaatatgctgtactttttccacTTTTTctgagtcaaagcat
atgtattttaaagaattgtgccttttTTTTTgaagttaccgtttt
gcagtatcctttccacacgctTTTTTctgagtcaaagcat
tggttgcatagttagcacatagtctTTTTTgaagttaccgtttt
cgttacaggttaagcttctaggttcTTTTTctgagtcaaagcat
attacacacagacaggatgtgcacTTTTTgaagttaccgtttt
agagcactgcacaacaagtcgaTTTTTctgagtcaaagcat
In certain embodiments, the target polynucleotide is an E6 and/or E7 sequence from low risk-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 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 (Tables 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 510 (Table 56).
cctctgcagtacttaacgtttttctgTTTTTctcttggaaagaaagt
gcacgtccagaattgacttatttgtTTTTTctcttggaaagaaagt
tttaatgaatcgcgccttgtcTTTTTctcttggaaagaaagt
cgacccttccaggtatcttgtaaTTTTTctcttggaaagaaagt
cctttaaggtagtatagtttccatgcaTTTTTctcttggaaagaaagt
gaggttccagctgtaaaacaatttTTTTTctcttggaaagaaagt
gcagacgtggaggcatttgcTTTTTgaagttaccgtttt
ccttgcacaactggtctatactttgtTTTTTctgagtcaaagcat
attgtgcataggaatgttgcactTTTTTgaagttaccgtttt
caaaacacgcataaaatttgcagTTTTTctgagtcaaagcat
acaaatggcacaggctgcaTTTTTgaagttaccgtttt
tgattgaccttaccttgtagttctaaTTTTTctgagtcaaagcat
ccgcgtagttaaaatgcctaaatTTTTTgaagttaccgtttt
ttcttcttccactgttactgcatatcTTTTTctgagtcaaagcat
ggcacaaatagcagcgtatcaTTTTTgaagttaccgtttt
cacgtggcacaatggtttgtTTTTTctgagtcaaagcat
caatgtaggcctacagggtcagTTTTTgaagttaccgtttt
tctgagctgtctaattgctcattgTTTTTctgagtcaaagcat
In other embodiments, the target polynucleotide is hepadnavirus 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, and/or 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).
ggggagWccgcgtaaagagatttttctcttggaaagaaagt
tgcRacgtgcagaggtgaatttttctcttggaaagaaagt
RagtccaagagtcctcttatgYaagactttttctcttggaaagaaagt
cagaggtgaaaaagttgcatgRtttttctcttggaaagaaagt
aMggRtcaatgtccatgcctttttctcttggaaagaaagt
tcagaaggcaaaaaMgagagtaacttttttctcttggaaagaaagt
atDgcttgcctKagtgcHgtatgtttttctcttggaaagaaagt
cggaagtgttgataagataggggtttttctcttggaaagaaagt
ctKcgtctgcgaggcgagtttttctcttggaaagaaagt
ggcagatgagaaggcacagactttttgaagttaccgtttt
gcgaagtgcacacggWcctttttctgagtcaaagcat
tcggtcgttgacattRcWgRtttttgaagttaccgtttt
cagtctttgaagtaKgcctcaaggtttttctgagtcaaagcat
gcctacagcctccYaRtacaaagactttttgaagttaccgtttt
tgMtggtgMRcaSaccaatttattttttctgagtcaaagcat
ggacatgWacaWgagatgatYaggtttttgaagttaccgtttt
cagcttggaggcttgaacagtRtttttctgagtcaaagcat
agactctaaggcYtcYcgatactttttgaagttaccgtttt
RtgaggtgaRcaatgYtcMggtttttctgagtcaaagcat
cctcKtcgtctaacaacagtagtYtctttttgaagttaccgtttt
cagcttggaggcttgaacagtRtttttctgagtcaaagcat
cgacgcggcgattgagaYtttttgaagttaccgtttt
attcccgagattgagatctYctgtttttctgagtcaaagcat
gWgtccaaggRatactaacattgagtttttgaagttaccgtttt
cccMgtaaaRtttcccaccttattttttctgagtcaaagcat
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 (e.g., at least two, three, four, or five) may comprise a target-hybridizing sequence selected from Table 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).
ttcatttcctccaatccccttatggTTTTTctcttggaaagaaagt
attgctgtgatatctttcatgttatcttgaTTTTTctcttggaaagaaagt
actgctaccagaattactttcccttctaaatgTTTTTctcttggaaagaaagt
cgctggtaaaattgctgccattgtctTTTTTctcttggaaagaaagt
ctccttgactttggggattgtagggaTTTTTctcttggaaagaaagt
ctgattccagagctgactaatttatctacttgTTTTTctgagtcaaagcatgaagttac
gccttatctatcccatctaaaaatagtatcttcTTTTTctgagtcaaagcatgaagttac
agattaaaatcactagccattgctctccaTTTTTctgagtcaaagcatgaagttac
ggctactatttcctttgctactataggtggcTTTTTctgagtcaaagcatgaagttac
gactacagtctacctgtccatgcatggcTTTTTctgagtcaaagcatgaagttac
ctgcttctatatagccactggctacatggTTTTTctgagtcaaagcatgaagttac
cctgccctgtttctgctgggataacttTTTTTctgagtcaaagcatgaagttac
gtgtgtactacttttactggccatcctccTTTTTctgagtcaaagcatgaagttac
ccaccaacaggctgctttaaatgcagTTTTTctgagtcaaagcatgaagttac
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).
tgcttctatatacccactggctacatgaactTTTTTctcttggaaagaaagt
caacaggcggccttgaccgcTTTTTctcttggaaagaaagt
tcctgcttgacccccgcccacTTTTTctcttggaaagaaagt
gcactatagtccccaatcccccctcTTTTTctcttggaaagaaagt
ctttccagaggagctttgctggtccTTTTTctcttggaaagaaagt
actcttatcttgtattactactgccccttcacTTTTTctcttggaaagaaagt
caatctagttgccatattcctggactacagtTTTTTctgagtcaaagcatgaagttac
gctaccaggataactatccttctaaatgtgtaTTTTTctgagtcaaagcatgaagttac
gccctgtctctgctgggatcacttcTTTTTctgagtcaaagcatgaagttac
tgtatgtattgtctttactggccatcttccTTTTTctgagtcaaagcatgaagttac
ttctttattcatagattctactactccttgactTTTTTctgagtcaaagcatgaagttac
gatctcttacctgtcctataattttctttaaTTTTTctgagtcaaagcatgaagttac
tttgtactgctgtcttaagatgttcagcctTTTTTctgagtcaaagcatgaagttac
ttcttttaaaattgtggatgaatactgccaTTTTTctgagtcaaagcatgaagttac
ctgttgctattatgtctattattctctcccctTTTTTctgagtcaaagcatgaagttac
aatttgtttttgtaattctttggtttgtatgtTTTTTctgagtcaaagcatgaagttac
tgtaatagacccgaaaattttgaatttttgtTTTTTctgagtcaaagcatgaagttac
gccatctgttttccataatccctaatgatTTTTTctgagtcaaagcatgaagttac
cctgtctacttgccatacaatcatcacctTTTTTctgagtcaaagcatgaagttac
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, 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).
ttctttagctctttattcattgattctactactccTTTTTctct
ttcccctgcactgtaccccccaatcTTTTTctcttggaaagaaa
gctgtccctgtaataaacccggaaattttTTTTTctcttggaaa
tgccccttcacctttccagagtagctTTTTTctcttggaaagaa
cgtcacctgccatctgttttccataatcTTTTTctcttggaaag
ttcagcttgatctcttacctgtcctatgatcTTTTTctgagtca
atactgccatttgtactgctgtcttaagatgTTTTTctgagtca
cccccttttcttttaaaattgtggatgaTTTTTctgagtcaaag
ttgtatgtctgttgctattatgtctattattctTTTTTctgagt
gaatttttgtaacttgtttttgtaattctttagtTTTTTctgag
ttgctggtcctttccaaactgggtctctTTTTTctgagtcaaag
ctacttttatttcactattgtcttgtatgactacTTTTTctgag
tcatcctgtctacctgccacacaatTTTTTctgagtcaaagcat
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 (NA), 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 (Tables 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 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).
ggttctattggaaaatgaaagaactttTTTTTctcttggaaaga
caaaatgagcaggggtcaggTTTTTctcttggaaagaaagt
tgccggtttcattgaagggTTTTTctcttggaaagaaagt
ccagcctcccatttcagaatatacTTTTTctcttggaaagaaa
acacccaagggtgctataaacaTTTTTctcttggaaagaaagt
caagccggaaatagcaataagaTTTTTctcttggaaagaaagt
aaaaggaaattcatacccaaagcTTTTTctcttggaaagaaagt
aaaggtgtaacggcagcatgtTTTTTctcttggaaagaaagt
ctcagtgtcatcatttgaaaggtttTTTTTctcttggaaagaaa
gggtaaatgtaacattgctggctTTTTTctcttggaaagaaagt
ggttctattggaaaatgaaagaactttTTTTTctcttggaaaga
caaaatgagcaggggtcaggTTTTTctcttggaaagaaagt
tgccggtttcattgaagggTTTTTctcttggaaagaaagt
ccagcctcccatttcagaatatacTTTTTctcttggaaagaaa
acacccaagggtgctataaacaTTTTTctcttggaaagaaagt
caagccggaaatagcaataagaTTTTTctcttggaaagaaagt
aaaaggaaattcatacccaaagcTTTTTctcttggaaagaaagt
aaaggtgtaacggcagcatgtTTTTTctcttggaaagaaagt
ctcagtgtcatcatttgaaaggtttTTTTTctcttggaaagaaa
ccagtaaactgattaactggttcttcaTTTTTctcttggaaaga
tggacttacaatgccgaactgttTTTTTgaagttaccgtttt
tgatgatggtttcctggacattTTTTTctgagtcaaagcat
ggtaaagagttcaaccacctggaTTTTTgaagttaccgtttt
aagatgaatacacagttcacagcagtaTTTTTctgagtcaaagc
cacacagaatgccattgacgagTTTTTgaagttaccgtttt
atatgcagccgacctgaagagTTTTTctgagtcaaagcat
tggatggtacggttatcaccatTTTTTgaagttaccgtttt
gggtggacagggatggtagaTTTTTctgagtcaaagcat
aggcctatttggggccatTTTTTgaagttaccgtttt
ggaatatcccgtctattcaatctagTTTTTctgagtcaaagcat
tgagactggccacaggattgaTTTTTgaagttaccgtttt
tccaaaatatgtaaaaagcacaaaatTTTTTctgagtcaaagc
cacgattgcaatacaacttgtcaaTTTTTgaagttaccgtttt
ggtattatcatttcagatacaccagtcTTTTTctgagtcaaagc
gtggtaccgagatatgcattcgTTTTTgaagttaccgtttt
cattcgaagcaactggaaatctaTTTTTctgagtcaaagcat
agtagagccgggagacaaaataaTTTTTgaagttaccgtttt
gggagaatgaactattactggacactTTTTTctgagtcaaagc
aatgcagatacatatgtttttgtggTTTTTgaagttaccgtttt
tgctgaccaacaaagtctctatcagTTTTTctgagtcaaagcat
ttctacaaaaatttaatatggctagttaaTTTTTgaagttaccg
cctcatgctggagcaaaaagcTTTTTctgagtcaaagcat
ggcccaatcatgactcgaacTTTTTgaagttaccgtttt
gagatattccccaagacaagttcatTTTTTctgagtcaaagcat
tgaggagctaagagagcaattgagTTTTTgaagttaccgtttt
tacccaggagatttcatcgattaTTTTTctgagtcaaagcat
cctagttcagacaatggaacgtgtTTTTTgaagttaccgtttt
catggtcctacattgtggaaacaTTTTTctgagtcaaagcat
tgaatcactctccacagcaagctTTTTTgaagttaccgtttt
ggatcctgggaaatccagagtgTTTTTctgagtcaaagcat
tggacttacaatgccgaactgttTTTTTgaagttaccgtttt
tgatgatggtttcctggacattTTTTTctgagtcaaagcat
ggtaaagagttcaaccacctggaTTTTTgaagttaccgtttt
aagatgaatacacagttcacagcagtaTTTTTctgagtcaaagc
cacacagaatgccattgacgagTTTTTgaagttaccgtttt
atatgcagccgacctgaagagTTTTTctgagtcaaagcat
tggatggtacggttatcaccatTTTTTgaagttaccgtttt
gggtggacagggatggtagaTTTTTctgagtcaaagcat
aggcctatttggggccatTTTTTgaagttaccgtttt
ggaatatcccgtctattcaatctagTTTTTctgagtcaaagcat
tgagactggccacaggattgaTTTTTgaagttaccgtttt
tccaaaatatgtaaaaagcacaaaatTTTTTctgagtcaaagc
cacgattgcaatacaacttgtcaaTTTTTgaagttaccgtttt
ggtattatcatttcagatacaccagtcTTTTTctgagtcaaagc
gtggtaccgagatatgcattcgTTTTTgaagttaccgtttt
cattcgaagcaactggaaatctaTTTTTctgagtcaaagcat
agtagagccgggagacaaaataaTTTTTgaagttaccgtttt
gggagaatgaactattactggacactTTTTTctgagtcaaagc
aatgcagatacatatgtttttgtggTTTTTgaagttaccgtttt
tgctgaccaacaaagtctctatcagTTTTTctgagtcaaagcat
ttctacaaaaatttaatatggctagttaaTTTTTgaagttaccg
cctcatgctggagcaaaaagcTTTTTctgagtcaaagcat
ggcccaatcatgactcgaacTTTTTgaagttaccgtttt
gagatattccccaagacaagttcatTTTTTctgagtcaaagcat
tgaggagctaagagagcaattgagTTTTTgaagttaccgtttt
tacccaggagatttcatcgattaTTTTTctgagtcaaagcat
cctagttcagacaatggaacgtgtTTTTTgaagttaccgtttt
catggtcctacattgtggaaacaTTTTTctgagtcaaagcat
tgaatcactctccacagcaagctTTTTTgaagttaccgtttt
ggatcctgggaaatccagagtgTTTTTctgagtcaaagcat
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 ID 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).
taacgccatggctagacgctttctgcgtgaTTTTTctcttggaa
gcgggttgatccaagaaaggacccggtcTTTTTctcttggaaag
ggcacgcccaaatctccaggcattgaTTTTTctcttggaaagaa
agacctcccggggcactcgcaaTTTTTctcttggaaagaaagt
cctaccctcgggctggcgagccTTTTTctcttggaaagaaagt
caccccaagccctcattgccatagagTTTTTctcttggaaagaa
cgagcggaatgtaccccatgagatcggTTTTTctcttggaaaga
ggtcctggaggctgcacgacactcatacTTTTTctgagtcaaag
ccgcagaccactatggctctcccgTTTTTctgagtcaaagcatg
gtcctggcaattccggtgtactcaccgTTTTTctgagtcaaagc
caacactactcggctagcagtctcgcgggTTTTTctgagtcaaa
caccctatcaggcagtaccacaaggccttTTTTTctgagtcaaa
ttcgtgctcatggtgcacggtctacgTTTTTctgagtcaaagca
ggtgttacgtttggtttttctttgaggtttagTTTTTctgagtc
cccctgcgcggcaacaggtaaactccacTTTTTctgagtcaaag
gtcgcgcgcacacccaacctggTTTTTctgagtcaaagcatgaa
ttggggataggttgtcgccttccacgaTTTTTctgagtcaaagc
acggggtgacaggagccatcctgccTTTTTctgagtcaaagcat
ccaaattgcgcgacctacgccggggTTTTTctgagtcaaagcat
aagccgcacgtgagggtatcgatgaccttacTTTTTctgagtca
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).
ccagtaaactgattaactggttcttcaTTTTTctcttggaaaga
gtgtatgttagcagcatttacaatcaTTTTTctcttggaaagaa
ccttttgcatggcaccattgTTTTTctcttggaaagaaagt
gcaagattatgtccagaaagcaaaTTTTTctcttggaaagaaa
gctgccttaagaagctggatgtTTTTTctcttggaaagaaagt
ggtttagcaccaaatatgcctgTTTTTctcttggaaagaaagt
caatctctgattgctcagtagtatcatTTTTTgaagttaccgtt
ggtgtaggttctggttgggctTTTTTctgagtcaaagcat
ggcaacattgtcagtaagttttaaataaTTTTTgaagttaccgt
tccttaacgatgtcaacacatttaatTTTTTctgagtcaaagc
gctacaccaccaccatgtttcagTTTTTgaagttaccgtttt
gttgccttgttgagtgcacctTTTTTctgagtcaaagcat
ccatttagcttaatgtaatcatcactctTTTTTgaagttaccgt
caagaccctcctactgtaagagggTTTTTctgagtcaaagcat
ccaacaacatgcagacacttcttaTTTTTgaagttaccgtttt
cctcacctgcatttaggttaggtTTTTTctgagtcaaagcat
tgtcctgtgaattgaaattttcatatTTTTTgaagttaccgtt
ctgacaacaatggtgcaagtaagaTTTTTctgagtcaaagcat
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. gonorrheae; Hemophilus influenzae; Actinomyces (e.g., Norcardia); Acinetobacter; Bacillaceae (e.g., Bacillus anthrasis); Bacteroides (e.g., Bacteroides fragilis); Blastomycosis; Bordetella (Bordetella pertusi); Borrelia (e.g., Borrelia burgdorferi); Brucella; Campylobacter; Chlamydia; Coccidioides; Corynebacterium (e.g., Corynebacterium diptheriae); E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli); Enterobacter (e.g. Enterobacter aerogenes); Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Serratia, Yersinia, Shigella); 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); Rickettsiaceae; 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).
The target polynucleotides from the bacteria can be a polynucleotide, DNA or a RNA (such as a ribosomal 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.
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).
accgctaaagcgctttccaTTTTTctcttggaaagaaagt
ccgcgggctcatcccacTTTTTctcttggaaagaaagt
tggccggacaccctctcaTTTTTctcttggaaagaaagt
agttaagccgtgagatttcacgTTTTTctcttggaaagaaagt
tcgcccgcacgctcacTTTTTctcttggaaagaaagt
cgccttggtaggccgtcaTTTTTgaagttaccgtttt
ggccggctacccgtcgtTTTTTctgagtcaaagcat
ggccgtatctcagtcccagtgTTTTTgaagttaccgtttt
gctgcctcccgtaggagtctgTTTTTctgagtcaaagcat
ccattgtgcaatattccccactTTTTTgaagttaccgtttt
gctgcatcaggcttgcgcTTTTTctgagtcaaagcat
tcccccacgcggcgtcTTTTTgaagttaccgtttt
tacaacccgaaggccgtcaTTTTTctgagtcaaagcat
tgcttcttctccacctaccgtcTTTTTgaagttaccgtttt
tggcacgtagttggccggTTTTTctgagtcaaagcat
ctacgtattaccgcggctgcTTTTTgaagttaccgtttt
ccggacaacgctcgcaccTTTTTctgagtcaaagcat
cgagctctttacgcccagtaattTTTTTgaagttaccgtttt
aacaacgcgacaaaccacctaTTTTTctgagtcaaagcat
tuberculosis
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).
ccccttacgtgttaccgcgTTTTTctcttggaaagaaagt
ccaggcttaccagtccgccTTTTTctcttggaaagaaagt
cctccgtgattctagaccagcaTTTTTctcttggaaagaaagt
ttcgccaccggtgttcttcTTTTTctcttggaaagaaagt
cgcacctcagcgtcaatcatTTTTTctcttggaaagaaagt
taatgcgttcgcgtcggTTTTTctcttggaaagaaagt
ggggcttattcgcacgactTTTTTgaagttaccgtttt
gctgctggcacgtaattagccTTTTTctgagtcaaagcat
aataattccgaacaacgctcgTTTTTgaagttaccgtttt
tgcatgccctttacgcccTTTTTctgagtcaaagcat
ttgagctcggggatttcacaTTTTTgaagttaccgtttt
gtacccagtgcagttcccaagTTTTTctgagtcaaagcat
cacttggaattccggtttccTTTTTgaagttaccgtttt
caaatatctacagattccacccctaTTTTTctgagtcaaagcat
tgttcgctccccacaccttTTTTTgaagttaccgtttt
tgtggactaccagggtatctaatccTTTTTctgagtcaaagcat
caacacctagtgtacatcgtttactgTTTTTgaagttaccgtt
cgccgagactcatgcccTTTTTctgagtcaaagcat
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 fumigatus, 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 brasiliensis); pneumocystis pneumonia (Pneumocystis jiroveci); sporotrichosis (Sporothrix schenckii); etc.
In one embodiment, the present invention provides a method for detecting fungal target polynucleotides from an Aspergillus, for example, Aspergillus fumigatus. A. fumigatus is a common, often lethal and difficult to diagnose cause of pathogenic disease in immuno-compromised 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 18S 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 Table 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).
actcgcggtgaggcggaTTTTTctcttggaaagaaagt
aaggtccagccggaccagtTTTTTctcttggaaagaaagt
catgaggttccccagaaggaTTTTTctcttggaaagaaagt
cagctgaatactgacgccccTTTTTctcttggaaagaaagt
gaaaacatccttggcgaatgTTTTTctcttggaaagaaagt
cgagcgggtcatcatagaaacTTTTTctcttggaaagaaagt
aggttcaactacgagctttttaactgTTTTTgaagttaccgtt
ccggccagccagacccaTTTTTctgagtcaaagcat
gcctgctttgaacactctaattttTTTTTgaagttaccgtttt
catgctaatgtattcgagcaaagTTTTTctgagtcaaagcat
cggtcctagaaaccaacaaaatagaTTTTTgaagttaccgtttt
cgactatccctattaatcattacggTTTTTctgagtcaaagcat
aaatccaagaatttcacctctgaTTTTTgaagttaccgtttt
ctttcgcagtagttagtcttcagcTTTTTctgagtcaaagcat
cctaactttcgttccctgattaatTTTTTgaagttaccgtttt
cggtatctgatcgtcttcgatccTTTTTctgagtcaaagcat
ggcatagtttatggttaagactacgaTTTTTgaagttaccgtt
accgcccgatccctagtcTTTTTctgagtcaaagcat
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).
aaaaagatggaccggccagTTTTTctcttggaaagaaagt
acccagaaggaaaggctcgTTTTTctcttggaaagaaagt
tggttcgccataaatggctTTTTTctcttggaaagaaagt
actgatacccccgaccgtcTTTTTctcttggaaagaaagt
aaacgtccttggtaaatgctttcTTTTTctcttggaaagaaagt
gtgccgattgcgtcaataaaaTTTTTctcttggaaagaaagt
gagctttttaactgcaacaactttaataTTTTTgaagttaccgt
ccaagcccaaggttcaactacTTTTTctgagtcaaagcat
gagcaaaggcctgctttgaaTTTTTgaagttaccgtttt
cctattctattattccatgctaatatattcTTTTTctgagtcaa
aaccaacaaaatagaaccataacgtTTTTTgaagttaccgtttt
cctattaatcattacgatggtcctagaTTTTTctgagtcaaagc
agaatttcacctctgacaactgaatTTTTTgaagttaccgtttt
gcagtagttagtcttcagtaaatccaTTTTTctgagtcaaagc
cctaactttcgttcttgattaatgaTTTTTgaagttaccgtttt
cggtatctgatcatcttcgatccTTTTTctgagtcaaagcat
ggcatagtttatggttaagactacgaTTTTTgaagttaccgtt
gaacaacaaccgatccctagtcTTTTTctgagtcaaagcat
In another embodiment, the fungal target polynucleotides are from Cryptococcus. In particular, the target polynucleotides are from Cryptococcus neoformans, a medically important species of the Crytococcus best known for causing a severe form of meningitis and meningoencephalitis 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 Cryptococcus 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).
gcctcgccagacctgaagttTTTTTctcttggaaagaaagt
gcactccgtgaggaggaccTTTTTctcttggaaagaaagt
ggttcccctgcacacccagTTTTTctcttggaaagaaagt
gcgattgcctgctttgaacacTTTTTctcttggaaagaaagt
cggcatcgtttactgttaagactaTTTTTctcttggaaagaaa
cgtgggccgatccctagtTTTTTctcttggaaagaaagt
gaggtaaggtccagcaagacagtTTTTTgaagttaccgtttt
taaagagcatacaggaccaccagTTTTTctgagtcaaagcat
tattattccatgctaatgtattcggTTTTTgaagttaccgtttt
aaatagaaccgcacgtcctattcTTTTTctgagtcaaagcat
cggcgatcctagaaaccaacaTTTTTgaagttaccgtttt
ccgaccgtccctattaatcattaTTTTTctgagtcaaagcat
ccgtcaatctaagaatttcacctctaTTTTTgaagttaccgtt
gctttcgcagttgttggtcttTTTTTctgagtcaaagcat
ccctaaccttcgttcttgatcaTTTTTgaagttaccgtttt
caacggtatctaatcgtttttgatcTTTTTctgagtcaaagcat
gccgacccagtcagagattgaTTTTTgaagttaccgtttt
caaagactttgatttctcgtaaggtTTTTTctgagtcaaagcat
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 fropica mexicana, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania chagasi); trypanosomiasis (Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense); toxoplasmosis (Toxoplasma gondii); schistosomiasis (Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Schistosoma mekongi, Schistosoma intercalatum); malaria (Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale); Amebiasis (Entamoeba histolytica); Babesiosis (Babesiosis microti); Cryptosporidiosis (Cryptosporidium parvum); Dientamoebiasis (Dientamoeba fragilis); Giardiasis (Giardia lamblia); Helminthiasis and Trichomonas (Trichomonas vaginalis).
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 target polynucleotide. In certain embodiments, the housekeeping gene polynucleotide is glyceraldehyde 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).
ggtcaatgaaggggtcattgatTTTTTctcttggaaagaaagt
ccttgacggtgccatggaatTTTTTctcttggaaagaaagt
cgccccacttgattttggaTTTTTctcttggaaagaaagt
gagatgatgacccttttggctcTTTTTctcttggaaagaaagt
ggagcagagagcgaagcggTTTTTgaagttaccgtttt
cggctgactgtcgaacaggaTTTTTctgagtcaaagcat
gagcgatgtggctcggcTTTTTgaagttaccgtttt
tcaccttccccatggtgtctTTTTTctgagtcaaagcat
ccaggcgcccaatacgacTTTTTgaagttaccgtttt
agttaaaagcagccctggtgaTTTTTctgagtcaaagcat
tggaacatgtaaaccatgtagttgaTTTTTgaagttaccgtttt
ttgccatgggtggaatcatatTTTTTctgagtcaaagcat
tgacaagcttcccgttctcagTTTTTgaagttaccgtttt
tggtgatgggatttccattgaTTTTTctgagtcaaagcat
gacgtactcagcgccagcatTTTTTgaagttaccgtttt
aagacgccagtggactccacTTTTTctgagtcaaagcat
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 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.
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 cell line having a particular characteristic of interest.
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.
In certain embodiments, the kit is configured such that the level of target polynucleotide and level of normlaization 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).
TABLE 96 shows a comparison of the invention with currently available HPV-tests.
The schematic in
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
The results show that the method of the invention using the probe set described for detecting HPV has a sensitivity of about 3000 copies, which, given the high specificity of the probe set, is sufficient for accurate HPV detection.
The results in TABLE 98 and
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 precancerous. CIN3 lesions represent severe neoplasia involving the full thickness of the mucosa and this class includes carcinoma in situ, a pre-invasive 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 grade.
The results show that this embodiment can detect as low as 25 copies. In addition,
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.
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.
TABLE 99 shows that the H1N1 assay does not cross-react with various strains of Influenza A, Influenza B or other respiratory virus.
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 show 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.
The results show detection measured by log relative light units (RLU). 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 (
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.
The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will be 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.
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.
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.
