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

Plasmids pUC-S and pUC-N were combined with human cDNA and used in each multiplex PCR reaction. Paragon Genomics' CleanPlex® multiplex PCR reagents and protocol were used. Briefly, a 10µl multiplex PCR reaction was made by combining 5X mPCR mix, 10X Pool 1 of the panel, plasmid pUC-S, pUC-N and cDNA. The reaction was run in a thermal cycler (95°C for 10min, then 98°C for 15sec, 60°C for 5min for 10 cycles), then terminated by the addition of 2µl of stop buffer. The reaction was then purified by 29µl of magnetic beads, followed by a secondary PCR with a pair of primers for 25 cycles. The secondary PCR added sample indexes and sequencing adapters, allowing for sequencing of the resulting products by high throughput sequencing. A final bead purification was performed after the secondary PCR, followed by library interrogation using a Bioanalyzer 2100 instrument with Agilent High Sensitivity DNA Kit (Agilent Technologies, Inc. Part# 5067-4626). The copyright holder for this preprint (which was not peer-reviewed) is the .