ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens

Emerg Microbes Infect. 2020 Dec;9(1):1259-1268. doi: 10.1080/22221751.2020.1772678.

Abstract

Quantitative real time PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load specimens and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 RNA from 77 patients, and compared with RT-PCR in terms of the diagnostic accuracy based on the results of follow-up survey. 26 patients of COVID-19 with negative RT-PCR reports were reported as positive by ddPCR. The sensitivity, specificity, PPV, NPV, negative likelihood ratio (NLR) and accuracy were improved from 40% (95% CI: 27-55%), 100% (95% CI: 54-100%), 100%, 16% (95% CI: 13-19%), 0.6 (95% CI: 0.48-0.75) and 47% (95% CI: 33-60%) for RT-PCR to 94% (95% CI: 83-99%), 100% (95% CI: 48-100%), 100%, 63% (95% CI: 36-83%), 0.06 (95% CI: 0.02-0.18), and 95% (95% CI: 84-99%) for ddPCR, respectively. Moreover, 6/14 (42.9%) convalescents were detected as positive by ddPCR at 5-12 days post discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the RT-PCR.

Keywords: RT-PCR; SARS-CoV-2; clinical detection; droplet digital PCR; false negative.

MeSH terms

  • Betacoronavirus / genetics*
  • COVID-19
  • Coronavirus Infections / diagnosis*
  • False Negative Reactions
  • Humans
  • Limit of Detection
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • SARS-CoV-2
  • Viral Load / methods

Substances

  • RNA, Viral

Grant support

This study was supported by Special Fund for COVID-19 Research of Wuhan University, China National Science and Technology Major Project [grant number 2018ZX10733403 and 2018YFA0900801], China NSFC grants [grant number 32041007], Wuhan COVID-19 Emergency Science and Technology Project [grant number 2020020201010012], the Fundamental Research Funds for the Central Universities and the National Mega Project on Major Infectious Disease Prevention [grant number 2017ZX10103005]. 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.