Reagents and methods for detecting HCV
The present disclosure relates to oligonucleotide sequences for amplification primers and their use in performing nucleic acid amplifications of HCV, in particular regions that encode the NS3 polypeptide. In some embodiments the primers are used in nested PCR methods for the detection or sequencing of HCV NS3. The oligonucleotide sequences are also provided assembled as kits that can be used to amplify and detect or sequence HCV NS3.
1. A method of performing a polymerase chain reaction (PCR) on an HCV NS3 1a target nucleic acid sequence in a sample comprising:
contacting the sample with a set of outer primers, wherein the set of outer primers comprises one or more forward primers and one or more reverse primers,
wherein the one or more forward primers comprise:
a primer comprising SEQ ID NO: 1 or an active fragment thereof,
a primer comprising SEQ ID NO: 2 or an active fragment thereof,
a primer comprising SEQ ID NO: 3 or an active fragment thereof,
a primer comprising SEQ ID NO: 4 or an active fragment thereof,
a primer comprising SEQ ID NO: 5 or an active fragment thereof, or
a combination thereof; and
wherein the one or more reverse primers comprise:
a primer comprising SEQ ID NO: 6 or an active fragment thereof,
a primer comprising SEQ ID NO: 7 or an active fragment thereof,
a primer comprising SEQ ID NO: 8 or an active fragment thereof,
a primer comprising SEQ ID NO: 9 or an active fragment thereof,
a primer comprising SEQ ID NO: 10 or an active fragment thereof, or
a combination thereof;
wherein an active fragment is 15 or more nucleotides in length; and submitting the resulting mixture to a first PCR.
2. The method of claim 1 , wherein the one or more forward primers comprise:
a primer comprising SEQ ID NO: 1 or an active fragment thereof,
a primer comprising SEQ ID NO: 2 or an active fragment thereof,
a primer comprising SEQ ID NO: 3 or an active fragment thereof,
a primer comprising SEQ ID NO: 4 or an active fragment thereof, and
a primer comprising SEQ ID NO: 5 or an active fragment thereof.
3. The method of claim 1 , wherein the one or more reverse primers comprise:
a primer comprising SEQ ID NO: 6 or an active fragment thereof,
a primer comprising SEQ ID NO: 7 or an active fragment thereof,
a primer comprising SEQ ID NO: 8 or an active fragment thereof,
a primer comprising SEQ ID NO: 9 or an active fragment thereof, and
a primer comprising SEQ ID NO: 10 or an active fragment thereof.
4. The method of claim 2 , wherein the one or more reverse primers comprise:
a primer comprising SEQ ID NO: 6 or an active fragment thereof,
a primer comprising SEQ ID NO: 7 or an active fragment thereof,
a primer comprising SEQ ID NO: 8 or an active fragment thereof,
a primer comprising SEQ ID NO: 9 or an active fragment thereof, and
a primer comprising SEQ ID NO: 10 or an active fragment thereof.
5. The method of claim 1 , further comprising contacting a product from the first PCR with a set of inner primers and submitting the resulting mixture to a second PCR.
6. The method of claim 5 , wherein at least some of the inner primers are labeled.
7. The method of claim 5 , wherein at least some of the inner primers are tagged with tag sequences.
8. The method of claim 7 , wherein the tag sequences are M13 tag sequences.
9. The method of claim 5 , further comprising sequencing a product from the second PCR to detect an HCV NS3 1a polymorphism.
10. The method of claim 5 , wherein the first PCR is a reverse transcription (RT) PCR.
11. The method of claim 10 , wherein the RT PCR is a kinetic PCR or kinetic RT PCR.
12. The method of claim 1 , wherein the sample comprises a primary sample obtained by methods selected from the group consisting of: biopsy, surgery, and collection of body fluid.
13. The method of claim 12 , wherein the sample further comprises a secondary sample comprising nucleic acids and, optionally, proteins derived from the primary sample.
14. The method of claim 1 , wherein the target nucleic acid is selected from the group consisting of: DNA and cDNA.
15. The method of claim 14 , wherein the sample further comprises RNA.
16. The method of claim 1 , wherein the one or more forward primers are selected from the group consisting of:
a primer comprising SEQ ID NO: 1,
a primer comprising SEQ ID NO: 2,
a primer comprising SEQ ID NO: 3,
a primer comprising SEQ ID NO: 4, and
a primer comprising SEQ ID NO: 5; and
wherein the one or more reverse primers are selected from the group consisting of:
a primer comprising SEQ ID NO: 6,
a primer comprising SEQ ID NO: 7,
a primer comprising SEQ ID NO: 8,
a primer comprising SEQ ID NO: 9, and
a primer comprising SEQ ID NO: 10.
17. A method of performing a polymerase chain reaction (PCR) on an HCV NS3 1a target nucleic acid sequence in a sample comprising:
contacting the sample with a set of outer primers, wherein the set of outer primers comprises one or more forward primers and one or more reverse primers,
wherein the one or more forward primers comprise one of:
a primer comprising SEQ ID NO: 1,
a primer comprising SEQ ID NO: 2,
a primer comprising SEQ ID NO: 3,
a primer comprising SEQ ID NO: 4, or
a primer comprising SEQ ID NO: 5; and
wherein the one or more reverse primers comprise one of:
a primer comprising SEQ ID NO: 6,
a primer comprising SEQ ID NO: 7,
a primer comprising SEQ ID NO: 8,
a primer comprising SEQ ID NO: 9, or
a primer comprising SEQ ID NO: 10; and
submitting the resulting mixture to a first PCR.