Reliance 1 step multiplex enzyme supermix

RT-qPCR was performed following an NA extraction-free approach, in which 50 μl of virus sample was first treated with 6.25 μl of proteinase K followed by a heat inactivation step and was then directly used as input. The 2019-nCov CDC EUA Kit (10006770, Integrated DNA Technologies) for detection of N1, N2, and ribonuclease P genes and a Reliance 1 step multiplex enzyme supermix (12010176, Bio-Rad) were used to prepare PCR cocktail including 1 μl of N1 probe, 1 μl of N2 probe, 6.25 μl of enzyme mix, 1.75 μl of nuclease-free water, and 15 μl of proteinase K–treated virus sample. The RT-qPCR was conducted on a Real-Time PCR Detection System (CFX384, Bio-Rad) following the protocol: 52°C for 10 min, 95°C for 2 min, and 45 cycles of 95°C for 10 s and 55°C for 30 s. The Ct value for the samples without detection signal was recorded as 45 for quantitative comparison. To obtain the lysed virus sample, viruses were incubated at 65°C for 30 min for antigen analysis by the QUIT SARS-CoV-2 system and treated with proteinase K for 5 min for NA analysis by RT-qPCR.

RT-qPCR was performed following a nucleic acid extraction-free approach, in which 50 μL of virus sample was first treated with 6.25 μL proteinase K followed by a heat inactivation step and was then directly used as input. A 2019-nCov CDC EUA Kit (10006770, Integrated DNA Technologies) and a Reliance 1 step multiplex enzyme supermix (12010176, Bio-Rad) were used to prepare PCR cocktail including 1 μL N1 probe, 1 μL N2 probe, 6.25 μL enzyme mix, 1.75 μL nuclease-free water, and 15 μL proteinase K-treated virus sample. The RT-qPCR was conducted on a Real-Time PCR Detection System (CFX384, Bio-Rad) following the protocol: 52 °C for 10 min, 95 °C for 2 min, and 45 cycles of 95 °C for 10 s and 55 °C for 30 s. The Ct value for the samples without detection signal was recorded as 45 for quantitative comparison. To obtain the lysed virus sample, viruses were incubated at 65 °C for 30 min for antigen analysis by QUIT SARS-CoV-2 system and treated with proteinase K for 5 min for nucleic acid analysis by RT-qPCR.