Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Expert Rev Mol Diagn. 2021 Jan;21(1):119-129. doi: 10.1080/14737159.2021.1865807. Epub 2020 Dec 30.


Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening.Methods: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system.Results: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug could not.Conclusion: Our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude-based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.

Keywords: COVID-19; RT-PCR; diagnosis; droplet digital PCR; multiplex assays; sars-CoV-2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antiviral Agents / pharmacology
  • COVID-19 / diagnosis*
  • COVID-19 Nucleic Acid Testing*
  • DNA Primers / genetics
  • False Positive Reactions
  • Humans
  • Limit of Detection
  • Multiplex Polymerase Chain Reaction / methods*
  • Pandemics
  • RNA, Viral / isolation & purification
  • Real-Time Polymerase Chain Reaction / methods
  • Reproducibility of Results
  • SARS-CoV-2 / isolation & purification*
  • Sensitivity and Specificity
  • Temperature
  • Viral Load / methods


  • Antiviral Agents
  • DNA Primers
  • RNA, Viral

Grant support

This research was funded by ‘Megaproject of Infectious Disease Control from Ministry of Health of China, grant number 2017ZX10302301-005’ and ‘Sino-Africa Joint Research Center, grant number SAJC201605.’