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 amplifying HCV NS3 in a sample comprising contacting a sample with a set of outer primers, wherein the set of outer primers comprises forward primers and reverse primers, the forward primers comprising: 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, and the reverse primers comprising 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; and submitting the resulting mixture to a first nucleic acid amplification reaction.
2 . The method of claim 1 , wherein the forward primers comprise a primer comprising SEQ ID NO: 11 or an active fragment thereof, a primer comprising SEQ ID NO: 12 or an active fragment thereof, a primer comprising SEQ ID NO: 13 or an active fragment thereof, a primer comprising SEQ ID NO: 14 or an active fragment thereof, and a primer comprising SEQ ID NO: 15 or an active fragment thereof, and the reverse primers comprise a primer comprising SEQ ID NO: 16 or an active fragment thereof, a primer comprising SEQ ID NO: 17 or an active fragment thereof, a primer comprising SEQ ID NO: 18 or an active fragment thereof, a primer comprising SEQ ID NO: 19 or an active fragment thereof, and a primer comprising SEQ ID NO: 20 or an active fragment thereof.