Genetic marker for detection of human papillomavirus
The invention provides compositions and methods for the differential detection of high risk forms of HPV from a urine sample provided by a patient. Specifically, the invention provides primers and probes that specifically recognize and bind sequences within the E 1 gene of HPV. Detection of high risk forms of HPV identify individuals at risk of developing or in the early stages of cervical carcinoma.
1. An oligonucleotide having the sequence 5′-CAGGCAGAATTAGAGRCAGC (SEQ ID NO:41 or 99), wherein R is A or G, and the oligonucleotide is optionally labeled.
2. The oligonucleotide according to claim 1 , wherein the oligonucleotide is fluorescently labeled.
3. The oligonucleotide according to claim 2 , wherein the oligonucleotide is XEN-HPV-FAM-F (SEQ ID NO:98).
4. A pair of oligonucleotide primers comprising a forward primer that is the oligonucleotide of claim 1 and a reverse primer having the sequence 5′-TCCACCACAWACTTTCGTTTTA (SEQ ID NO:42 or 100), wherein W is T or A.
5. The oligonucleotide primers according to claim 4 , wherein the forward primer is fluorescently labeled.
6. The oligonucleotide primers according to claim 5 , wherein the forward primer is XEN-HPV-FAM-F (SEQ ID NO:98) and the reverse primer is XEN-HPV-R (SEQ ID NO:100).
7. A kit comprising the oligonucleotide according to claim 1 .
8. A kit comprising the oligonucleotide according to claim 2 .
9. A kit comprising the oligonucleotide according to claim 3 .
10. A kit comprising the oligonucleotide primers according to 4 .
11. A kit comprising the oligonucleotide primers according to 5 .
12. A kit comprising the oligonucleotide primers according to 6 .
13. An oligonucleotide primer having the sequence 5′-TCCACCACAWACTTTCGTTTTA (SEQ ID NO:42 or 100), wherein W is T or A.
14. A method of detecting human papillomavirus (HPV) infection in a patient, the method comprising:
detecting one or more sequences of the E1 gene of HPV in a urine sample obtained from a patient,
wherein said detecting comprises hybridization of said one or more sequences to the oligonucleotide of claim 1 and wherein detection of one or more sequences of the E1 gene of HPV indicates the presence of at least one HPV infection in a patient.
15. The method of claim 14 , wherein said amplifying comprises a technique selected from the polymerase chain reaction (PCR); nested primer PCR; Real Time PCR; NA hybridization; Cyclic Probe Reaction; Single-Strand Conformation Polymorphism (SSCP); Strand Displacement Amplification (STA); and Restriction Fragment Length Polymorphism (RFLP).
16. The method of claim 14 , wherein said detecting comprises
(a) amplifying nucleic acids, from said urine sample obtained from a patient, with said oligonucleotide as a forward primer to produce amplified E1 gene sequences; and
(b) detecting the amplified E1 gene sequences.
17. The method of claim 16 , wherein said amplifying further comprises an oligonucleotide having the sequence 5′-TCCACCACAWACTTTCGTTTTA (SEQ ID NO:42 or 100), wherein W is T or A.
18. The method of claim 17 , wherein said amplifying comprises XEN-HPV-FAM-F (SEQ ID NO:98) and XEN-HPV-R (SEQ ID NO:100) as primers.
19. The method of claim 14 , wherein said nucleic acids comprise transrenal DNA.
20. The method of claim 14 , further comprising reducing nucleic acid degradation in the urine sample.
21. The method of claim 20 , wherein reducing nucleic acid degradation comprises inhibiting nuclease activity by increased pH, increased salt concentration, heat inactivation, or by treating said urine sample with a compound selected from the group consisting of: ethylenediaminetetraacetic acid, guanidine-HCl guanidine isothiocyanate, N-lauroylsarcosine, and sodium dodecylsulphate.
22. The method of claim 14 , further comprising substantially isolating said nucleic acids from the urine sample before said amplifying.
23. The method of claim 22 , wherein said isolating is by precipitation or using a solid adsorbent material.
24. The method of claim 14 , further comprising the step of quantifying said amplified E1 gene sequences.
25. The method of claim 14 , wherein said detecting further comprises the extension of said oligonucleotide after hybridization.