Life (Oct 2021)

A Multiallelic Molecular Beacon-Based Real-Time RT-PCR Assay for the Detection of SARS-CoV-2

  • Andreas C. Chrysostomou,
  • Johana Hezka Rodosthenous,
  • Cicek Topcu,
  • Christina Papa,
  • Antonia Aristokleous,
  • Georgia Stathi,
  • Christina Christodoulou,
  • Christina Eleftheriou,
  • Dora C. Stylianou,
  • Leondios G. Kostrikis

DOI
https://doi.org/10.3390/life11111146
Journal volume & issue
Vol. 11, no. 11
p. 1146

Abstract

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Emerging infectious viruses have led to global advances in the development of specific and sensitive detection techniques. Viruses have an inherent potential to easily mutate, presenting major hurdles for diagnostics and requiring methods capable of detecting genetically diverse viral strains. One such infectious agent is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in December 2019 and has resulted in the global coronavirus disease 2019 (COVID-19) pandemic. This study presents a real-time reverse transcription PCR (RT-PCR) detection assay for SARS-CoV-2, taking into account its intrinsic polymorphic nature that arises due to genetic drift and recombination, as well as the possibility of continuous and multiple introductions of genetically nonidentical strains into the human population. This advance was achieved by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV-2 S, E, M, and N genes. These were applied to create a simple and reproducible real-time RT-PCR assay, which was validated using external quality control panels (QCMD: CVOP20, WHO: SARS-CoV-2-EQAP-01) and clinical samples. This assay was designed for high target detection accuracy and specificity and can also be readily adapted for the detection of other emerging and rapidly mutating pathogens.

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