High sensitivity detection of coronavirus SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method

Chenyu Li; David N. Debruyne; Julia Spencer; Vidushi Kapoor; Lily Y. Liu; Bo Zhou; Lucie Lee; Rounak Feigelman; Grayson Burdon; Jeffrey Liu; Alejandra Oliva; Adam Borcherding; Hongdong Tan; Alexander E. Urban; Guoying Liu; Zhitong Liu

Several RT-PCR methods for detecting SARS-CoV-2 have been reported to date 15, [19] [20] [21] . Among them, two groups reported the detection of 4-5 copies of the virus 19, 21 . To investigate the opportunity for further improvement on the sensitivity of RT-PCR, we built a mathematical model to estimate the limit of detection (LOD) for SARS-CoV-2. The reported RT-PCR amplicon lengths are around 78-158bp, and the SARS-CoV-2 genome is 29,903bp (NC_045512.2). Thus, in this mathematical modeling, we chose 100bp amplicon length and 30kb SARS-CoV-2 genome size for the estimation. With the assumption of 99% RT-PCR efficiency 20 , we found that RT-PCR assays could detect 4.8 copies of SARS-CoV-2 at 95% probability ( Fig. 1A) , which is consistent with the experimental results obtained in a previous report 19 . In this model, the probability of RT-PCR assays to detect one copy of SARS-CoV-2 is only 26% (Supplemental Fig. 1 ). This finding may explain, at least in part, the low reported 47-56% detection rates of SARS-CoV-2 in positive samples by RT-PCR 17, 22 . We further found that the LOD appears to be independent of the virus genome size. For genomes of 4 to 100kb, the detection limit remains 4.8 copies at 95% probability.