One step reverse transcription pcr

Plasma was separated from whole blood. Viral RNA was extracted from 140 μl of plasma by using the QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA, United States) according to the manufacturer’s instructions, and was then subjected to nested polymerase chain reaction (PCR) to amplify the fragments of the E1E2 (H77: 933–2060) and NS5B (H77: 8340–9233). The primers and conditions of PCR reaction were described previously [28 (link)]. The first PCR reaction was performed using One Step reverse transcription PCR (Takara, Dalian, China). The second PCR reaction was performed using 2×Taq PCR MasterMix (Tiangen, Beijing, China). PCR products were sent to ZIXIBIO Co. (Beijing, China) for sequencing using an ABI 3730XL automated DNA sequencer (Applied Biosystems, Carlsbad, USA).

HPgV-1 RNA was isolated from 200-µL plasma samples using the High Pure Viral RNA Kit according to the procedure described in the manual (Roche, Basel, Switzerland)). Then, the 5’-UTR (U36380:119-497) and E2 (U36380:950-1844) sequences were amplified by nested PCR; the PCR primers (supplement Table S1) and conditions were as reported in previous study [7 (link),9 (link)]. Owing to the high degree of conservation and amplification efficiency of the 5’-UTR, this region was used to evaluate the HPgV-1 infection rate.
The E2 region was used to determine the HPgV-1 genotype, as this sequence could analyze the different genotypes with the same consistency as the complete genome [7 (link),8 (link),9 (link)]. The first PCR reaction was performed using One Step reverse transcription PCR (Takara, Dalian, China) and the second using 2 × Taq PCR MasterMix (Tiangen, Beijing, China). The PCR products were firstly detected by agarose gel (1.0%) electrophoresis and visualized under ultraviolet (UV) illumination for the presence of an 895-nucleotide band and then purified by using a DNA purification kit (Tiangen, Beijing, China); subsequently, the purified products were sent to Shenzhen Invitrogen Biotechnology Co., Ltd. (Shenzhen, Guangdong, China) for sequencing by using an ABI 3730XL automated DNA sequencer (Applied Biosystems, Carlsbad, CA, USA).