Nested PCR for HIV gene sequencing

RNA samples were directly subjected to nested polymerase chain reactions (PCR) to generate fragments of gag (HXB2: 781–1861; encoding portions of p17 and p24), pol (HXB2: 2147–3462; encoding the protease and the first 299 residues of reverse transcriptase) and env (HXB2: 7002–7541, encoding the V3∼V4 region). The first PCR reaction was performed using One Step reverse transcription PCR Kit (Takara, Dalian, China). The second PCR reaction was performed using 2×Taq PCR Master Mix (Tiangen, Beijing, China). The details of primers and PCR reaction conditions were previously described [19 (link)]. The products were analyzed using 1% agarose gel electrophoresis. Positive samples were separately sent to ZIXIBIO Co. (Beijing, China) for sequencing using an ABI 3730XL automated DNA sequencer (Applied Biosystems, Carlsbad, USA) with the following primers: gag: GUX/GDX; env: 33F/48R; pol: PROS3, RTAS, RTB, PROC1S, and RT20S3. The details of sequencing primers were previously described [19 (link)].