Abstract: Objective Although the epidemic of coronavirus disease 2019 (COVID-19) has been controlled in China, large-scale testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still needed. Therefore, it is necessary to establish a method that can detect the new coronavirus on a large scale and improve the sensitivity and accuracy of the new coronavirus detection. Methods The dilution of mixed multiple samples could cause the low copy number of the virus and result in the inaccurate detection. The nested PCR coupled with qRT-PCR was designed to improve the sensitivity and accuracy of detection. The copy number of the tested sample was greatly increased by external primer PCR. The N gene sequence recommended by the National Center for Disease Control and Prevention (CDC) was then detected by fluorescent quantitative PCR. Results The false-positive rate of detection was reduced, and the detection sensitivity was increased by 3 orders of magnitude on the original basis, and a single copy of the virus template could be stably detected. Conclusion This Nested-qRT-PCR can greatly improve the sensitivity of new coronavirus detection and provide a feasible method for large-scale detection of mixed multiple samples. 

Key words: coronavirus, nucleic acid detection, qPCR, sensitivity, false-positive rate