Multiplex RT-PCR for Respiratory Viruses

Ribonucleic acid (RNA) was extracted using MagMax-96 viral RNA isolation kit as per manufacturer’s instruction [18 (link)]. Real-time Reverse Transcription PCR (qRT-PCR) were performed for the following respiratory viruses: influenza A virus, influenza B virus, respiratory syncytial virus (RSV) A and B, human metapneumovirus (hMPV), Para-influenza virus (PIV) 1,2,3 and 4, rhinovirus, adeno virus and corona virus (HCoV – 229E, OC43, HKU1) using the Invitrogen Superscript III one step quantitative RT-PCR kit (Invitrogen, Thermo Fischer Scientific, USA) [19 (link)]. This RT-PCR assay was carried out on ABI 7500 machine (Applied BiosystemsInc, USA). PCR reaction mixture was prepared for 25 µl that comprise of 10 µmol of forward and reverse primers, 5 µmol of Taqman probe, 12.5 µl of 2 × buffer, 0.5 µl of superscript^TMIII enzyme and 5 µl of nucleic acid template. Thermal cycling conditions for these qRT-PCR tests consisted of 45 cycles reaction with initial denaturation at 94⁰C for 5 min, denaturation at 94⁰C for 15 s, annealing at 55⁰C for 30 s [18 (link), 19 (link)].