Detection of SARS-CoV-2 RNA by multiplex RT-qPCR
E. Kudo, B. Israelow, C. Vogels, P. Lu, A. L. Wyllie, M. Tokuyama, A. Venkataraman, D. E. Brackney, I. Ott, M. Petrone, R. Earnest, S. Lapidus, C. Muenker, A. Moore, A. Casanovas-Massana, Yale IMPACT Research Team, S. Omer, C. Dela Cruz, S. Farhadian, A. Ko, N. Grubaugh, A. Iwasaki
Abstract
The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. To simplify testing and reduce the volume of required reagents, we developed 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 CDC, 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 singleplex assay adapted for research purposes. Low copies (>500 copies / reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current singleplex diagnostics by saving reagents, costs, time and labor.
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