Quantitative SARS-CoV-2 RNA Evaluation

For assessment of viral replication at 24 and 48 h post-infection, RNA from cell culture supernatants was extracted by using Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega, Fitchburg, WI, USA) through the Maxwell^® RSC Instrument (Promega, Fitchburg, WI, USA). Viral RNA was quantified as previously described [20 (link)]. Briefly, single-step, real-time, RT-qPCR (GoTaq^® 1-Step RT-qPCR, Promega, Fitchburg, WI, USA) and the 2019-nCoV CDC qPCR Probe Assay kit with probes targeting SARS-CoV-2 nucleocapsid (N) gene and the human RNase P gene as an internal control (IDT, Coralville, IA, USA) were used on a CFX96 instrument (Bio-Rad, Hercules, CA, USA). Absolute viral copy number quantification was performed by generating a standard curve from the quantified 2019-nCoV_N-positive Plasmid Control (IDT, Coralville, IA, USA). A cycle threshold (Ct) value of <40 was considered positive, based on CDC guidelines.