A culture-free method for rapidly and accurately quantifying active SARS-CoV-2

Determining the quantity of active virus is the most important basis to judge the risk of virus infection, which usually relies on the virus median tissue culture infectious dose (TCID₅₀) assay performed in a biosafety level 3 laboratory within 5-7 days. We have developed a culture-free method for rapid and accurate quantification of active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by targeting subgenomic RNA (sgRNA) based on reverse transcription digital PCR (RT-dPCR). The dynamic range of quantitative assays for sgRNA-N and sgRNA-E by RT-dPCR was investigated, and the result showed that the limits of detection (LoD) and quantification (LoQ) were 2 copies/reaction and 10 copies/reaction, respectively. The delta strain (NMDC60042793) of SARS-CoV-2 was cultured at an average titer of 10^6.13 TCID₅₀/mL and used to evaluate the developed quantification method. Copy number concentrations of the cultured SARS-CoV-2 sgRNA and genomic RNA (gRNA) gave excellent linearity (R² = 0.9999) with SARS-CoV-2 titers in the range from 500 to 10⁵ TCID₅₀/mL. Validation of 63 positive clinical samples further proves that the quantification of sgRNA-N by RT-dPCR is more sensitive for active virus quantitative detection. It is notable that we can infer the active virus titer through quantification of SARS-CoV-2 sgRNA based on the linear relationship in a biosafety level 2 laboratory within 3 h. It can be used to timely and effectively identify infectious patients and reduce unnecessary isolation especially when a large number of COVID-19 infected people impose a burden on medical resources.