A new SYBR Green real-time PCR to detect SARS-CoV-2

Sci Rep. 2021 Jan 26;11(1):2224. doi: 10.1038/s41598-021-81245-0.

Abstract

Phylogenetic analysis has demonstrated that the etiologic agent of the 2020 pandemic outbreak is a betacoronavirus named SARS-CoV-2. For public health interventions, a diagnostic test with high sensitivity and specificity is required. The gold standard protocol for diagnosis by the Word Health Organization (WHO) is RT-PCR. To detect low viral loads and perform large-scale screening, a low-cost diagnostic test is necessary. Here, we developed a cost-effective test capable of detecting SARS-CoV-2. We validated an auxiliary protocol for molecular diagnosis with the SYBR Green RT-PCR methodology to successfully screen negative cases of SARS-CoV-2. Our results revealed a set of primers with high specificity and no homology with other viruses from the Coronovideae family or human respiratory tract pathogenic viruses, presenting with complementarity only for rhinoviruses/enteroviruses and Legionella spp. Optimization of the annealing temperature and polymerization time led to a high specificity in the PCR products. We have developed a more affordable and swift methodology for negative SARS-CoV-2 screening. This methodology can be applied on a large scale to soften panic and economic burden through guidance for isolation strategies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzothiazoles
  • COVID-19 / diagnosis*
  • COVID-19 Nucleic Acid Testing / methods*
  • DNA, Single-Stranded
  • Diamines
  • Enterovirus
  • Genome, Viral
  • Humans
  • Organic Chemicals*
  • Phylogeny
  • Polymerase Chain Reaction
  • Quinolines
  • Real-Time Polymerase Chain Reaction / methods*
  • Rhinovirus
  • SARS-CoV-2*
  • Sensitivity and Specificity
  • Temperature
  • Viral Load

Substances

  • Benzothiazoles
  • DNA, Single-Stranded
  • Diamines
  • Organic Chemicals
  • Quinolines
  • SYBR Green I