Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant

Abstract

Introduction: The SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477 N substitution in this RBD.

Materials and methods: We aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant.

Results: All 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93 %) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 2018/3960 (51%) patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6 %) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR.

Discussion: Our in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in about half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.

Keywords: Covid-19; Diagnosis; Marseille-4; Molecular epidemiology; SARS-CoV-2; Variant; qPCR.

Fig. 1

Three-dimensional structure of the SARS-CoV-2 spike protein showing amino acid substitutions and deletions for major SARS-CoV-2 variants including the Marseille-4 variant. Structure prediction was performed using the Phyre2 web portal (http://www.sbg.bio.ic.ac.uk/∼phyre2/html/page.cgi?id=index) and visualized using the Pymol tool v.1.8 (https://pymol.org/2/). Amino acid substitutions and deletions are showed with a country flag for the variants first detected in UK (20I/501Y.V1), South Africa (20 H/501Y.V2) and Brazil (20 J/501Y.V3), or with the label “Marseille-4.

Fig. 2

Number and proportion of Marseille-4 variants detected by qPCR in respiratory samples from patients diagnosed with SARS-CoV-2 in our institute. The graph shows the weekly numbers of patients tested by our Marseille-4 specific qPCR assay (grey bars) and the weekly numbers (red bars) and proportions (red curve) of positive tests in January 2021. Systematic testing of RNA extracts obtained from nasopharyngeal samples of patients newly-diagnosed with SARS-CoV-2 was implemented in our institute from January 1 st, 2021 using our Marseille-4 specific qPCR assay (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).