SARS-CoV-2 qRT-PCR Detection Protocol

Total RNA was extracted using a MagNA Pure 96 External lysis buffer (Roche, Switzerland), and qRT-PCR reactions were performed using STANDARD M nCoV Real-Time Detection kit (M-NCOV-01, SD Biosensor, KOREA), which targeted regions of envelope (E) and RNA-dependent RNA polymerase (RdRp) [14 (link)] according to the manufacturer’s protocol. Briefly, PCR were run at an Applied Biosystems 7500 Real-Time PCR instrument system in a volume of 31 μL containing a 10 μL sample, a 14 μL 2019-nCoV reaction solution, 6 μL RTase mix, 0.5 μL ROX, and 0.5 μL internal control. The PCR conditions were as following: 15 min at 50 °C for reverse transcription, 3 min at 95 °C for initial denaturation, 5 cycles of 5 s at 95 °C and 40 s at 60 °C for pre-amplification, and 40 cycles of 5 s at 95 °C and 40 s at 60 °C for amplification. Viral RNA was shown as a cycle threshold (Ct) value that is inversely proportional to the original relative expression level of the target gene.