Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR

The ongoing COVID-19 pandemic has generated a global health crisis that needs well management of not only patients but also environments to reduce SARS-CoV-2 transmission. The gold standard RT-qPCR method is sensitive and rapid to detect SARS-CoV-2 nucleic acid, but does not answer if PCR-positive samples contain infectious virions. To circumvent this problem, we report an SDS-propidium monoazide (PMA) assisted RT-qPCR method that enables rapid discrimination of live and dead SARS-CoV-2 within 3 h. PMA, a photo-reactive dye, can react with viral RNA released or inside inactivated SARS-CoV-2 virions under assistance of 0.005% SDS, but not viral RNA inside live virions. Formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, RT-qPCR detection of heat-inactivated SARS-CoV-2 resulted in larger than 9 Ct value differences between PMA-treated and PMA-free groups, while less than 0.5 Ct differences were observed in the detection of infectious SARS-CoV-2 ranging from 20 to 5148 viral particles. Using a cutoff Ct difference of 8.6, this method could differentiate as low as 8 PFU live viruses in the mixtures of live and heat-inactivated virions. Further experiments showed that this method could successfully monitor the natural inactivation process of SARS-CoV-2 on plastic surfaces during storage with comparable results to the gold standard plaque assay. We believe that the culture-free method established here could be used for rapid and convenient determination of infectious SARS-CoV-2 virions in PCR-positive samples, which will facilitate better control of SARS-CoV-2 transmission.