SARS-CoV-2 RNA Detection by RT-PCR

Following inactivation, RNA from one aliquot per condition per virus isolate and negative control was immediately extracted with the QIAamp Viral RNA Mini Kit (Qiagen) and stored at −80 °C until further processing. Real-time RT-PCR assays for SARS-CoV-2 RNA detection were performed in duplicate using the Charité Virologie algorithm (Berlin, Germany) to detect both E and RdRp genes [9 (link)]. In brief, real-time RT-PCR was performed using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen) on the CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA) ABI. Each 25 µl reaction mixture contained 5 µl of RNA, 3.1 µl of RNase-free water, 12.5 µl of 2X PCR buffer, 1 µl of SuperScriptTM III RT/Platinum Taq Mix, 0.5 ul of each 10 µM forward and reverse primer, and 0.25 µl of probe (E_Sarbeco_P1 or RdRP_SARSr-P2) using the following thermal cycling conditions; 10 min at 55 °C for reverse transcription, 3 min at 94 °C for PCR initial activation, and 45 cycles of 15 s at 94 °C and 30 s at 58 °C.