Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA

PLoS One. 2020 Jul 24;15(7):e0236564. doi: 10.1371/journal.pone.0236564. eCollection 2020.

Abstract

To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65°C for 10 minutes along with the use of TaqPath™ 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x103 copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR CT values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, p = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.

MeSH terms

  • Betacoronavirus
  • COVID-19
  • COVID-19 Testing
  • Clinical Laboratory Techniques / methods*
  • Coronavirus Infections / diagnosis*
  • Humans
  • Nasopharynx / virology
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • RNA, Viral / isolation & purification*
  • Real-Time Polymerase Chain Reaction*
  • Reproducibility of Results
  • SARS-CoV-2
  • Sensitivity and Specificity
  • Specimen Handling / methods*

Substances

  • RNA, Viral

Grants and funding

The authors received no specific funding for this work.