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

PLoS Biol. 2020 Oct 7;18(10):e3000867. doi: 10.1371/journal.pbio.3000867. eCollection 2020 Oct.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Betacoronavirus / genetics*
  • Betacoronavirus / pathogenicity
  • COVID-19
  • COVID-19 Testing
  • Case-Control Studies
  • Clinical Laboratory Techniques / methods*
  • Clinical Laboratory Techniques / standards
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / virology
  • DNA Primers / standards
  • HEK293 Cells
  • Humans
  • Limit of Detection
  • Multiplex Polymerase Chain Reaction / standards*
  • Nasopharynx / virology
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / virology
  • RNA, Viral / genetics*
  • Reagent Kits, Diagnostic / standards*
  • Reverse Transcriptase Polymerase Chain Reaction / standards*
  • SARS-CoV-2
  • United States


  • DNA Primers
  • RNA, Viral
  • Reagent Kits, Diagnostic