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

Two plasmids, containing the full sequence of S and N genes of SARS-CoV-2, respectively, were used to validate our multiplex PCR method. 28 amplicons are expected to be amplified within our 172amplicon panel. To simulate the use of real clinical samples, these two plasmids were spiked into the cDNA made from human total RNA. The copy number of each plasmid was determined by droplet-based digital PCR in QX200 from Bio-Rad® 45 . The two plasmids were diluted from approximately 9,000 copies to below one copy, and were amplified in multiplex PCR reactions. The library peaks of expected sizes were obtained from 8,900 to 2.8 copies of plasmids ( Fig. 2A) . Quantification of peaks demonstrated a wide dynamic range from 1 to about 1,000 copies of plasmids (Fig. 2B) . The yield of the libraries started to saturate when the copy number was 1,700. It is possible that the saturation point could be even lower when all of the 172 amplicons are amplified from positive clinical samples, and the library peak could be observed with even fewer copies of virus. In contrast, the detected quantities of a single target on N gene by RT-PCR rapidly dropped when using 2.85 copies (Fig. 2B and Supplemental Fig. 3) .