SARS-CoV-2 Detection via One-Step RT-qPCR

Five microlitre of total RNA was used for one-step cDNA synthesis and RT-qPCR reaction in a total volume of 20 µL consisting of 20× Primer-Probe Mix for SARS-CoV-2 Detection (#PPM, Diagnolita) [11 (link)], 4× TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific (TFS), Waltham, MA, USA), and RNase-free water. For cDNA synthesis, reaction mixes were incubated at 50°C for 5 min, and the subsequent RT-qPCR runs consisted of enzyme activation at 95°C for 20 s and 40 cycles of 95°C for 3 s and 60°C for 30 s. RT-qPCR reactions were run on the QuantStudio 5 Real-Time PCR System by using QuantStudio Design & Analysis Software v1.4.1 (both from Applied Biosystems, TFS).