Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets

Emerg Microbes Infect. 2020 Dec;9(1):1175-1179. doi: 10.1080/22221751.2020.1772679.

Abstract

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.

Keywords: SARS-CoV-2; diagnosis; digital PCR; false negative; false positive; real time PCR.

Publication types

  • Letter

MeSH terms

  • Betacoronavirus / genetics*
  • COVID-19
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / virology*
  • DNA Primers
  • DNA Probes
  • Humans
  • Multiplex Polymerase Chain Reaction* / methods
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / virology*
  • Real-Time Polymerase Chain Reaction* / methods
  • Real-Time Polymerase Chain Reaction* / standards
  • SARS-CoV-2
  • Sensitivity and Specificity
  • Viral Load

Substances

  • DNA Primers
  • DNA Probes

Grants and funding

This study was supported by Special Fund for COVID-19 Research of Wuhan University, China National Science and Technology Major Project [grant numbers 2018ZX10733403 and 2018YFA0900801], China NSFC grants [grant number 32041007], Wuhan COVID-19 Emergency Science and Technology Project [grant number 2020020201010012] and the Fundamental Research Funds for the Central Universities. The research was designed, conducted, analysed, and interpreted by the authors entirely independently of the funding sources. The researchers confirm their independence from funders and sponsors.