Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays

Authors:

Mathieu Durand1, Philippe Thibault1, Simon Lévesque2,3, Ariane Brault4, Alex Carignan2, Louis Valiquette2, Philippe Martin2 and Simon Labbé4

doi: 10.15698/mic2022.01.767
Volume 9, pp. 1 to 20, published 25/11/2021.

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Affiliations:

1 Plateforme RNomique et de Génomique Fonctionnelle, Université de Sherbrooke, Sherbrooke, QC, Canada.

2 Département de Microbiologie et d’Infectiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada.

3 Laboratoire de Microbiologie, Centre Intégré Universitaire de Santé et de Services Sociaux (CIUSSS) de l’Estrie, Centre Hospitalier Universitaire de Sherbrooke (CHUS), Sherbrooke, QC, Canada.

4 Département de Biochimie et de Génomique Fonctionnelle, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada.

Keywords: 

COVID-19, SARS-CoV-2, genetic variants, real-time TaqMan reverse transcription PCR assays, molecular diagnostics, locked nucleic acid (LNA)

Corresponding Author(s):

Simon Labbé, Département de Biochimie et de Génomique Fonctionnelle, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada; Simon.Labbe@USherbrooke.ca

Conflict of interest statement:

The authors declare that they have no conflict of inter-est with the content of this article.

Please cite this article as:

Mathieu Durand, Philippe Thibault, Simon Lévesque, Ariane Brault, Alex Carignan, Louis Valiquette, Philippe Martin and Si-mon Labbé (2021). Detection of severe acute respirato-ry syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays. Microbial Cell 9(1): 1-20. doi: 10.15698/mic2022.01.767

© 2021 Durand et al. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduc-tion in any medium, provided the original author and source are acknowledged.

Abstract:

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected £ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.