A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions

Serafeim C Chaintoutis; Taxiarchis Chassalevris; George Tsiolas; Sofia Balaska; Ioannis Vlatakis; Evangelia Mouchtaropoulou; Victoria I Siarkou; Areti Tychala; Dimitris Koutsioulis; Lemonia Skoura; Anagnostis Argiriou; Chrysostomos I Dovas

doi:10.1016/j.jviromet.2021.114242

A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions

J Virol Methods. 2021 Oct:296:114242. doi: 10.1016/j.jviromet.2021.114242. Epub 2021 Jul 16.

Affiliations

¹ Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 11 Stavrou Voutyra str., 54627, Thessaloniki, Greece.
² Institute of Applied Biosciences, Centre of Research and Technology Hellas, Thermi, 57001, Thessaloniki, Greece.
³ Department of Microbiology, AHEPA University Hospital, Medical School, Faculty of Health Sciences, Aristotle University of Thessaloniki, 1 Stilponos Kyriakidi str., 54636, Thessaloniki, Greece.
⁴ EnzyQuest PC, Science and Technology Park of Crete, 100 Nikolaou Plastira str., Vassilika Vouton, 70013, Heraklion, Greece.
⁵ Laboratory of Microbiology and Infectious Diseases, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, University Campus, 54134, Thessaloniki, Greece.
⁶ Institute of Applied Biosciences, Centre of Research and Technology Hellas, Thermi, 57001, Thessaloniki, Greece; Department of Food Science and Nutrition, University of the Aegean, 81400, Myrina, Lemnos, Greece.
⁷ Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 11 Stavrou Voutyra str., 54627, Thessaloniki, Greece. Electronic address: dovas@vet.auth.gr.

Abstract

The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94 % and a linear range of over 5 log₁₀ copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs.

Keywords: Mutation of concern; Real-time RT-PCR; SARS-CoV-2; Spike protein; Typing.

MeSH terms

Amino Acid Substitution
COVID-19 / diagnosis
COVID-19 / virology*
COVID-19 Testing
Humans
Microbiological Techniques
Mutation*
Real-Time Polymerase Chain Reaction / methods*
Reverse Transcriptase Polymerase Chain Reaction / methods*
SARS-CoV-2 / classification
SARS-CoV-2 / genetics*
SARS-CoV-2 / isolation & purification
Spike Glycoprotein, Coronavirus / genetics*

Substances

Spike Glycoprotein, Coronavirus
spike protein, SARS-CoV-2