Detection of SARS-CoV-2 RNA by multiplex RT-qPCR

Kudo, Eriko; Israelow, Benjamin; Vogels, Chantal B. F.; Lu, Peiwen; Wyllie, Anne L.; Tokuyama, Maria; Venkataraman, Arvind; Brackney, Doug E.; Ott, Isabel M.; Petrone, Mary E.; Earnest, Rebecca; Lapidus, Sarah; Muenker, M. Catherine; Moore, Adam J.; Casanovas-Massana, Arnau; Omer, Saad B.; Dela Cruz, Charles S.; Farhadian, Shelli F.; Ko, Albert I.; Grubaugh, Nathan D.; Iwasaki, Akiko

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor.