Moloney murine leukaemia virus m mlv reverse transcriptase

The extracted nucleic acids from the CEMP assay with Ct ≤ 20 determined by the DAAN kits or ≤ Ct 30 determined by the BioGerm kits for SARS-CoV-2 nucleic acid detection were used as templates to synthesise cDNA using a conventional two-step reverse transcription reaction with random primers according to the manufacturer’s instructions. Moloney Murine Leukaemia virus (M-MLV) reverse transcriptase (200 U/μL) (Invitrogen, USA), Ribonuclease Inhibitor (50 U/μl, TransGen Biotech, China), and dNTP (10 mM, TransGen Biotech, China) were added separately to reaction mixtures.
To obtain the spike gene sequences for SARS-CoV-2 subtyping of Omicron, 5 μL cDNA obtained by reverse transcription was used as a template in a round of PCR using forward (S1:5’-AAT CTT AGG GAA TTT GTG TT-3’) and reverse (S2:5’-AGT ACT ACT ACT CTG TAT GG-3’) primers to get 976 bp products. All PCRs were performed in a 25 µL final volume mixture according to the kits 2 × EasyTaq® PCR SuperMix and manufacturer’s instructions (TransGen Biotech, China). Amplified products were visualised by electrophoresis on 2% agarose gels stained with ethidium bromide. The amplified products were sequenced by SinoGenoMax (Beijing, China).

Sample preparation, viral RNA extraction and c-DNA synthesis were performed as previously described [12 ,[15] (link), [16] (link), [17] (link),19 (link)]. Briefly, nasal or oropharyngeal swabs(Copan Diagnostics, Murrieta, United States) were sent to the National Influenza Centre, where 3 mL of cell culture medium [minimum essential medium (MEM) with N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) buffer with 5,000U/mL PenStrep)] were added to wash out the attached viruses. RNA was extracted from 200 µL sample material, employing the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche, Mannheim, Germany) or the MagNA Pure 24 Total NA Isolation Kit and eluting in 50 µL buffer. For c-DNA synthesis in a total volume of 40 µL, 25µL RNA, random hexamer primers and 200U Moloney murine leukaemia virus (M-MLV) Reverse Transcriptase (Thermo Fisher Scientific, Waltham, US) underwent reverse transcription under the following thermocycling conditions: 42°C (5 min), 37°C (30 min) and 95°C (5 min). c-DNA was diluted 1:1 with H₂O for downstream PCR assays.