Detection of HPV
View Patent ↗The present invention provides compositions and methods for the detection and characterization of HPV sequences. More particularly, the present invention provides compositions, methods and kits for using invasive cleavage structure assays (e.g. the INVADER assay) to screen nucleic acid samples, e.g., from patients, for the presence of any one of a collection of HPV sequences. The present invention also provides compositions, methods and kits for screening sets of HPV sequences in a single reaction container.
1. A method for detecting the presence or absence of a HPV target nucleic acid in a sample, wherein said HPV target nucleic acid is from one or more strains in a set of strains of HPV, comprising:
a) providing:
i) a sample suspected of containing a HPV target nucleic acid;
ii) a composition comprising a mixture of different oligonucleotide probe sets, wherein:
A) each of said oligonucleotide probe sets comprises a first oligonucleotide and a second oligonucleotide that in combination form an invasive cleavage structure with target nucleic acid from at least two different strains of HPV in said set of strains of HPV;
B) each of said oligonucleotide probe sets forms an invasive cleavage structure with target nucleic acid from fewer than all of the different strains in said set of strains of HPV; and
C) at least two of said probe sets in said mixture are configured to form an invasive cleavage structure with target nucleic acid from each different strain of HPV in said set of strains of HPV; and
iii) a 5′ nuclease;
b) exposing said sample to said composition under reaction conditions in which, if HPV target nucleic acid from one or more strains in said set of strains is present, at least two of said oligonucleotide probe sets hybridize to said HPV target nucleic acid to form at least two invasive cleavage structures, and in which said at least two invasive cleavage structures are cleaved with said 5′ nuclease; and
c) detecting cleavage of said invasive cleavage structures, wherein detection of cleavage of said invasive cleavage structures is indicative of the presence of HPV target nucleic acid from at least one strain of said set of strains of HPV in said sample.
2. The method of claim 1 , wherein said detection of cleavage is indicative of the presence of HPV target nucleic acid from at least one strain from the group of strains consisting of strains 16, 16Ty2, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 58iso, 59, 66, 67, 68, 68var, 69, 70, and 82.
3. The method of claim 1 , wherein said mixture detects HPV strains 51, 56, and 66.
4. The method of claim 1 , wherein said mixture detects HPV strains 39, 68, 68var, 45, 59 and 18.
5. The method of claim 1 , wherein said mixture detects HPV strains 33, 52, 58, 31, 35 and 16.
6. The method of claim 1 , wherein said mixture comprises the group of oligonucleotides consisting of SEQ ID NOS. 47, 51, 52, 79, and 80.
7. The method of claim 1 , wherein said mixture comprises the group of oligonucleotides consisting of SEQ ID NOS. 37, 38, 94, 95, 127, 129 and 142.
8. The method of claim 1 , wherein said mixture comprises the group of oligonucleotides consisting of SEQ ID NOS. 12, 13, 37, 38, 47, 51, 52, 79-80, 91, 94, 127, 129, and 142.
9. The method of claim 1 , wherein said 5′ nuclease is a FEN-1 endonuclease.
10. The method of claim 1 , wherein said 5′ nuclease is thermostable.
11. The method of claim 1 , wherein said HPV target nucleic acid is amplified prior to said exposure step.
12. The method of claim 1 , wherein said mixture of oligonucleotide probe sets further comprises a pair of oligonucleotide primers configured to amplify a sequence contained in said HPV target nucleic acid in a polymerase chain reaction.
13. The method of claim 1 , wherein said HPV target nucleic acid is a synthetic nucleic acid.
14. The method of claim 1 , wherein said HPV target nucleic acid is genomic HPV nucleic acid.
15. The method of claim 1 , wherein two or more mixtures of oligonucleotide probes are used together to detect said HPV target nucleic acid.
16. The method of claim 1 , wherein two or more of said mixtures of oligonucleotide probes are used together to detect all high-risk HPV strains.
17. The method of claim 1 , wherein said sample is from the group consisting of cervical cells, cervical secretions, epithelial cells, respiratory secretions, urethral cells, cells of the anogenital region, urine, saliva and biopsy tissue.
18. The method of claim 1 , wherein said second oligonucleotides comprise a detectable label.
19. The method of claim 1 , wherein cleavage of said invasive cleavage structures cleaves said second oligonucleotide to produce a cleaved 5′ region of said second oligonucleotide, wherein said providing step further comprises providing an oligonucleotide FRET cassette, wherein said oligonucleotide FRET cassette comprises a set of interactive labels, wherein said oligonucleotide FRET cassette is configured to form a second invasive cleavage structure with said cleaved 5′ region of said second oligonucleotide.
20. The method of claim 19 , wherein said cleaving further comprises cleaving said second invasive cleavage structure.
21. The method of claim 19 , wherein said detecting comprises detecting and/or measuring a signal produced from one or more members of a set of interactive labels.