A fast RT-qPCR system significantly shortens the time for SARS-CoV-2 nucleic acid test.

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to global development. Rapid and accurate diagnosis is critical for containing the pandemic and treating patients in time. As the gold standard for SARS-CoV-2 diagnosis, the qualitative reverse transcription-PCR (RT-qPCR) test has long been criticized for its long detection time. In this study, we optimized the primers and probes targeting SARS-CoV-2 ORF1ab and N gene designed by the Chinese Center for Disease Control and Preventions (CDC) to increase their Tm values to meet the optimal elongation temperature of Taq DNA polymerase, thus greatly shortened the elongation time. The higher elongation temperature in turn narrowed the temperature range of the reaction and saved more time. In addition, by shortening the distance between the fluorophore at the 5' end and the quencher in the middle we got a probe with higher signal-to-noise ratio. Finally, by using all these measures and optimized RT-qPCR program we successfully reduced the time (nucleic acid extraction step is not included) for nucleic acid test from 74 min to 26 min.