One step reverse transcription pcr kit

The viral nucleic acid was directly extracted from the clinical specimens using the Tianlong nucleic acid extraction kit (Tianlong Biotechnology, Xian, China) according to the manufacturer’s instructions. The samples were screened for human respiratory pathogens, including 16 viruses and 11 bacteria using multiplex real-time reverse transcriptase PCR (RT-PCR) with the TaqMan low-density array (TLDA) kit (Thermo Fisher Scientific Inc., Waltham, USA). The subtypes of HRSV were further identified by in-house real-time RT-PCR (20 (link)). The second hypervariable region (HVR2) of the G gene of HRSV B subtype was amplified using a one-step reverse transcription-PCR kit (TaKaRa Biotechnology, Dalian, China) and the primer pair GPB/F1 (21 (link)). The reaction conditions, as well as the purification and sequencing protocols, were as described previously (21 (link), 22 (link)).

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)].