Method for detection of high risk 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 E1 gene of HPV. Detection of high risk forms of HPV identifies individuals at risk of developing or in the early stages of cervical carcinoma.
1. A method of detecting a high risk human papillomavirus (HPV) infection in a patient, comprising:
(a) obtaining a urine sample from a patient; and
(b) detecting one or more sequences of the El gene of HPV in said urine sample by amplifying DNA in the urine sample using a forward primer having the sequence 5′-CAGGCAGAATTAGAGRCAGC (SEQ ID NO:41 or 99), wherein R is A or G, and detecting the amplification product;
wherein detecting one or more sequences of the E1 gene of HPV indicates the presence of at least one high risk HPV in the patient.
2. The method of claim 1 , wherein said DNA is transrenal DNA.
3. The method of claim 1 , wherein said amplifying comprises a technique selected from polymerase chain reaction (PCR); nested primer PCR; Real Time PCR; Cyclic Probe Reaction; Single-Strand Conformation Polymorphism (SSCP); Strand Displacement Amplification (STA); and Restriction Fragment Length Polymorphism (RFLP).
4. The method of claim 1 , further comprising quantifying the high risk HPV in the sample.
5. A kit for the performance of the method of claim 1 , said kit comprising a container for one or more sequences of the E1 gene of HPV in said urine sample and one or more oligonucleotides for detecting said one or more sequences, wherein said one or more oligonucleotides comprises the sequence 5′-CAGGCAGAATTAGAGRCAGC (SEQ ID NO:41 or 99), wherein R is A or G.
6. The method if claim 1 , wherein the forward primer is fluorescently labeled.
7. The method of claim 6 , wherein the forward primer is XEN-HPV-FAM-F (SEQ ID NO:98).
8. The method of claim 1 , wherein the amplifying further comprises using a reverse primer having the sequence 5′-TCCACCACAWACTTTCGTTTTA (SEQ ID NO:42 or 100), wherein W is T or A.
9. The method of claim 8 , wherein the reverse primer is XEN-HPV-R (SEQ ID NO:100).
10. The method of claim 9 , wherein the forward primer is XEN-HPV-FAM-F (SEQ ID NO:98).
11. The method of claim 1 , further comprising determining the genotype of the high risk HPV detected.
12. The method of claim 11 , wherein the genotype of the high risk HPV is determined using probes specific for each type of high risk HPV.
13. The method of claim 11 , wherein the genotype of the high risk HPV is determined using primer pairs that amplify each type of high risk HPV.
14. The method of claim 13 , wherein primers in the primer pairs that amplify each type of high risk HPV comprise at least one of SEQ ID NOs:16-40, or 43-59.
15. The method of claim 11 , wherein the genotype of the high risk HPV is determined using sequencing.