Quantitative SARS-CoV-2 RNA Detection

Viral RNA was extracted from 140 μL of cell supernatant using a QIAamp^® Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and stored at −80 °C until it was used. Quantitative analysis of purified RNA was performed using the in-house real-time RT-PCR protocol described by Skittrall [70 (link)], targeting a 222-base region within the RNA-dependent RNA polymerase (RdRp) region of the SARS-CoV-2 1ab gene. A standard curve was generated using duplicate serial dilutions (10⁸–10¹) of the SARS-CoV-2 NSP12 VersaClone cDNA plasmid (R&D Systems, Minneapolis, MN, USA; catalog no. RDC3140), propagated in TOP10 Chemically Competent E. coli cells (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) and quantified in an Eppendorf BioPhotometer (Hamburg, Germany) (ηg/μL) after purification using a QIAprep Spin Miniprep Kit (QIAGEN) according to the manufacturer’s instructions. Reactions were run on an Applied Biosystems 7500 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA).