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

Mathieu Durand; Philippe Thibault; Simon Lévesque; Ariane Brault; Alex Carignan; Louis Valiquette; Philippe Martin; Simon Labbé

doi:10.15698/mic2022.01.767

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

Microb Cell. 2021 Nov 25;9(1):1-20. doi: 10.15698/mic2022.01.767. eCollection 2022 Jan 3.

Authors

Mathieu Durand¹, Philippe Thibault¹, Simon Lévesque^{2

3}, Ariane Brault⁴, Alex Carignan², Louis Valiquette², Philippe Martin², Simon Labbé⁴

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.

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.

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

Grants and funding

We are grateful to Dr. Gilles Dupuis for critical reading of the manuscript and for his valuable comments. We thank Dr. Hugues Charest for his communication with respect to the N LSPQ primer-probe set and its utilization in rRT-PCR assays.