Comparison of Digital PCR and Quantitative PCR with Various SARS-CoV-2 Primer-Probe Sets

J Microbiol Biotechnol. 2021 Mar 28;31(3):358-367. doi: 10.4014/jmb.2009.09006.


The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

Keywords: COVID-19; SARS-CoV-2; droplet digital PCR (ddPCR); envelope protein gene; nucleocapsid protein gene; reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

MeSH terms

  • Animals
  • COVID-19 / diagnosis*
  • COVID-19 / virology
  • Cell Line
  • Chlorocebus aethiops
  • Gene Dosage / genetics
  • Humans
  • Nucleic Acid Probes / genetics*
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • SARS-CoV-2 / genetics*
  • Sensitivity and Specificity
  • Vero Cells


  • Nucleic Acid Probes
  • RNA, Viral