Detection of SARS-CoV-2, RSV, Influenza A and B
RNA was extracted using Zymo quick DNA/RNA extraction kit (Zymo Research, Irvine, USA) according to the manufacturer’s protocols. Briefly, 400µl of each sample was extracted and eluted using 50µl of elution buffer. RT-qPCR was done using Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Seegene Inc, Seoul, South Korea) on Bio-Rad CFX96 (Bio-Rad Laboratories, Hercules, USA). This multiplex assay can detect SARS-CoV-2 (N gene, RdRP gene and S gene), Influenza A, Influenza B and Respiratory syncytial virus simultaneously. 5µl of extracted RNA was added to 15µl of mastermix for each reaction and amplification was performed at 50°C for 20 mins, 95°C for 15 mins, 2 cycles of 95°C for 10 secs, 60°C for 40 secs, 72°C for 20 secs, 41 cycles of 95°C for 10 secs followed by fluorescence detection at 60°C for 15 secs and 72°C for 10 secs. All runs were performed together with relevant controls to ensure validity. The results were exported to Excel (Office 365) and interpreted using Seegene Viewer (Seegene Inc, Seoul, South Korea).