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

One way to elevate the sensitivity is to amplify multiple targets on the same virus genome in a multiplex PCR reaction, thereby increasing the frequency of occurrence in the mathematical model. Amplifying multiple targets has the advantage of potentially detecting fragments of degraded virus genome while withstanding sequencing variations, thus allowing for the detection of upcoming mutants. The amplification efficiency of multiplex PCR is critical for LOD. We estimated that the efficiency of our multiplex PCR technology is about 26% by using Unique Molecular Identifier (UMI)-labeled primers to count the amplified products after NGS sequencing (Supplemental Fig. 2 and Supplemental Table 1 ). However, the amplification efficiency could be lower, and not all amplicons amplified successfully if the template used is one single strand of cDNA. Thus, more amplicons are potentially required for multiplex PCR to detect limited copies of viruses.