Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs

Int J Mol Sci. 2020 Jun 20;21(12):4396. doi: 10.3390/ijms21124396.


The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

Keywords: COVID-19; SARS-CoV-2; coronavirus; qRT-PCR detection; test protocol.

Publication types

  • Evaluation Study

MeSH terms

  • Animals
  • Betacoronavirus / genetics
  • COVID-19 Testing
  • Caco-2 Cells
  • Chlorocebus aethiops
  • Clinical Laboratory Techniques / economics
  • Clinical Laboratory Techniques / methods*
  • Clinical Laboratory Techniques / standards
  • Coronavirus Infections / diagnosis
  • Coronavirus Infections / economics
  • Coronavirus M Proteins
  • Costs and Cost Analysis
  • Humans
  • RNA, Viral / chemistry
  • RNA, Viral / genetics
  • Reverse Transcriptase Polymerase Chain Reaction / economics
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards
  • SARS-CoV-2
  • Sensitivity and Specificity
  • Vero Cells
  • Viral Matrix Proteins / genetics


  • Coronavirus M Proteins
  • RNA, Viral
  • Viral Matrix Proteins
  • membrane protein, SARS-CoV-2