Development of a rapid viability RT-PCR (RV-RT-PCR) method to detect infectious SARS-CoV-2 from swabs

Since the rapid onset of the COVID-19 pandemic, its causative virus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), continues to spread and increase the number of fatalities. To expedite studies on understanding potential surface transmission of the virus and to aid environmental epidemiological investigations, we developed a rapid viability reverse transcriptase PCR (RV-RT-PCR) method that detects viable (infectious) SARS-CoV-2 from swab samples in <1 day compared to several days required by current gold-standard cell-culture-based methods. The method integrates cell-culture-based viral enrichment in a 96-well plate format with gene-specific RT-PCR-based analysis before and after sample incubation to determine the cycle threshold (C_T) difference (ΔC_T). An algorithm based on ΔC_T ≥ 6 representing ∼ 2-log or more increase in SARS-CoV-2 RNA following enrichment determines the presence of infectious virus. The RV-RT-PCR method with 2-hr viral infection and 9-hr post-infection incubation periods includes ultrafiltration to concentrate virions, resulting in detection of <50 SARS-CoV-2 virions in swab samples in 17 h (for a batch of 12 swabs), compared to days typically required by the cell-culture-based method. The SARS-CoV-2 RV-RT-PCR method may also be useful in clinical sample analysis and antiviral drug testing, and could serve as a model for developing rapid methods for other viruses of concern.