Method of determining and confirming the presence of an HPV in a sample

• US 9,605,303 B2
• Filed: 01/28/2011
• Issued: 03/28/2017
• Est. Priority Date: 01/29/2010
• Status: Expired due to Fees

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: Nazarenko, Irina
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US13/016,004
• Publication Number:
  US 20110294111A1
• Time in Patent Office: 2,251 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/708 for papilloma

Abstract

Methods are provided for genotyping a target nucleic acid in a sample. In various aspects, the methods comprise generating nucleic acid hybrids between probes specific for the genotypes of interest and the target nucleic acid and detecting hybridization in the sample. In other aspects, the methods comprise using multi-probe mixtures to reduce the volume of sample necessary to determine the genotype of the target nucleic acid.

Claims

1. A method for genotyping a target nucleic acid in a sample is provided comprising:

(a) generating a first detection mixture by a method comprising contacting a portion of the sample with a first probe set, wherein the first probe set comprises a nucleic acid probe specific for a first genotype of the target nucleic acid and a nucleic acid probe specific for a second genotype of the target nucleic acid, but does not comprise a nucleic acid probe specific for a third genotype of the target nucleic acid;

(b) generating a second detection mixture by a method comprising contacting a portion of the sample with a second probe set, wherein the second probe set comprises a nucleic acid probe specific for the second genotype of the target nucleic acid and a nucleic acid probe specific for the third genotype of the target nucleic acid, but does not comprise a nucleic acid probe specific for the first genotype of the target nucleic acid; and

(c) treating the first and second detection mixtures under conditions wherein the nucleic acid probes hybridize specifically to the first, second, and/or third genotype of the target nucleic acid; and

(d) detecting hybridization of the nucleic acid probe to the target nucleic acid, wherein;

(i) hybridization in the first detection mixture, but not the second detection mixture indicates that the sample comprises the first genotype of the target nucleic acid;

(ii) hybridization in the second detection mixture, but not the first detection mixture, indicates that the sample comprises the third genotype of the target nucleic acid; and

(iii) hybridization in the first detection mixture and the second detection mixture indicates that the sample comprises the second genotype of the target nucleic acid.

2. The method of claim 1 wherein the target nucleic acid is an HPV nucleic acid and the first, second, and third genotypes of the target nucleic acid are selected from the group consisting of HPV2, HPV3, HPV6, HPV10, HPV 11, HPV16, HPV18, HPV26, HPV27, HPV28, HPV29, HPV30, HPV31, HPV32, HPV33, HPV34, HPV35, HPV39, HPV42, HPV45, HPV51, HPV52, HPV53, HPV54, HPV56, HPV57, HPV58, HPV59, HPV64, HPV66, HPV67, HPV68, HPV69, HPV70, HPV73, HPV82, HPV84, HPV85, HPV86, HPV87, and HPV94.

3. The method of claim 2 wherein the first, second, and third genotypes of the target nucleic acid are HPV16, HPV18, and HPV45.

4. The method of claim 1 wherein the sample is a clinical sample preserved in a liquid based cytology medium.

5. The method of claim 4 wherein the liquid based cytology medium is selected from the group consisting of PRESERVCYT and SUREPATH.

6. The method of claim 1 wherein hybridization of the nucleic acid probe forms a DNA:

RNA hybrid.

7. The method of claim 6 wherein DNA:

RNA hybrid is detected by a method comprising contacting the DNA;

RNA hybrid with a DNA;

RNA-specific antibody.

8. The method of claim 1 wherein the target nucleic acid is first identified as being the first, second, or third genotypes of the target nucleic acid by a method comprising amplifying the target nucleic acid with a consensus primer capable of amplifying at least a portion of each of the first, second, and third genotypes of the target nucleic acid.

9. The method of claim 8 wherein the target nucleic acid is amplified by a method comprising qualitative PCR.

10. The method of claim 1 wherein the target nucleic acid is first identified as being the first, second, or third genotypes of the target nucleic acid by a method comprising hybridizing a consensus probe to the target nucleic acid, wherein the consensus probe is specific for each of the first, second, and third genotypes of the target nucleic acid.

11. The method of claim 10 wherein hybridization of the consensus probe to the first, second, and/or third HPV nucleic acids generates a DNA:

RNA hybrid.

12. The method of claim 11 wherein DNA:

RNA hybrid is detected by a method comprising contacting the DNA;

RNA hybrid with a DNA;

RNA-specific antibody.

13. The method of claim 6 wherein DNA:

RNA hybrid is detected by a method comprising contacting the DNA;

RNA hybrid with a DNA;

RNA-specific antibody.

14. The method of claim 1 comprising:

(1) performing a qualitative PCR reaction on the sample to obtain an initial indication of the presence of an high risk-HPV genotype;

(2) performing a hybrid capture assay to confirm a determination of the presence of the HR-HPV; and

(3) conducting a PS test to genotype the high risk-HPV determined to be present in the sample.