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, 2,018/3,960 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 more than 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.

Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France

“…It seems obvious that targeting one or two mutations by PCR cannot precisely detect the variants if they are not widespread. The PCR approach remains highly effective on well-targeted variants, as we have determined in our local setting by successively implementing real-time RT-PCR specific to the N501Y-harbouring variants and of the Marseille-4 and Marseille-1 variants [ 47 , 48 , 49 ]. It is likely that, in the future, molecular screening based on PCR will have to benefit from a multiplexing strategy capable of targeting several mutations simultaneously and will have to continuously adapt to the constant increasing viral diversity and complexity of mutation patterns of circulating variants.…”

High individual heterogeneity of neutralizing activities against the original strain and 9 different variants of SARS-CoV-2

“…Eleven patients were infected by spike D614G-harbouring B lineage strains that spread during the rst wave of COVID-19 infections in France, 9 were infected by the Marseille-4/B.1.160 variant, 10 were infected by the UK/B.1.1.7 variant and 12 were infected by the South African/B.1.351 variant. Direct genotyping from respiratory samples was performed under previously described conditions [7]. Along with these sera, 13 sera from vaccinated individuals were also selected.…”

High individual heterogeneity of neutralizing activities against the original 4 strain and 9 different variants of SARS-CoV-2

“…A total of 55 human serum samples were included as part of a sero-epidemiological study that is being performed in our laboratory, and the patients' sera included 42 sera obtained from convalescent patients within 3 weeks to 5 months after a documented COVID-19 infection (Supporting Table S1 and Supporting Table S2). Eleven patients were infected by spike D614G-harbouring B lineage strains that spread during the first wave of COVID-19 infections in respiratory samples was performed under previously described conditions [7]. Along with these sera, 13 sera from vaccinated individuals were also selected.…”