key: cord-0725147-2c65npwd
authors: Bedotto, M.; Fournier, P.-E.; Houhamdi, L.; LEVASSEUR, A.; Delerce, J.; Pinault, L.; Padane, A.; CHAMIEH, A.; TISSOT-DUPONT, H.; BROUQUI, P.; Sokhna, C.; Azar, E.; Saile, R.; Mboup, S.; Bitam, I.; Colson, P.; Raoult, D.
title: Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant
date: 2021-02-08
journal: nan
DOI: 10.1101/2021.02.03.21250823
sha: ec2215e4d3620b04658ac6b82bb7a3399ce78e19
doc_id: 725147
cord_uid: 2c65npwd

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 S477N 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, 1,585/2,889 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.

Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR. 54

Discussion. Our in-house qPCR system was found reliable to detect specifically the 55 Marseille-4 variant and allowed estimating it is involved in more than half of our SARS-CoV-56 2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-57

CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical 58 characterics based on comprehensive sets of data. 59 60 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) substitution in the spike RBD and were reported in the UK and in South Africa, as highly 72 transmissible, and in Brazil, respectively. In our geographical area we detected 10 viral 73 variants since June 2020 [1] . One of them, we named Marseille-4, harbors a S477N 74 substitution in the spike RBD that has been associated with an improved binding affinity to 75 ACE2 [6] and a broad resistance to monoclonal neutralizing antibodies [7] . It predominates in 76

Marseille since August 2020, has been reported to spread in Europe since early summer and 77 was classified as the Nextstrain 20A.EU2 lineage [1, 2] . The continuous emergence of new 78 SARS-CoV-2 variants, including some of substantial concern regarding their transmissibility 79 and their possible ability to evade immune responses [8] [9] [10] , warrants to set up strategies for 80 their detection and surveillance. SARS-CoV-2 incidence is currently substantial in several 81 countries including France, and in our institute we for instance diagnose >100 new cases 82 daily. Therefore, alternative strategies to sequencing are useful for variant screening. We 83 aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay 84 that specifically detects the SARS-CoV-2 Marseille-4 variant. 85 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Our in-house qPCR system was found reliable to detect specifically the Marseille-4 129 variant and allowed estimating it is involved in more than half of our SARS-CoV-2 diagnoses 130 since December 2020. This assay is currently routinely used in our clinical microbiology and 131 virology laboratory to screen systematically all samples found SARS-CoV-2-positive using 132 the first-line qPCR diagnosis assay, which allows the real-time classification of viral strains in 133 more than half of the diagnoses (Figure 2 ). In case of negativity of this Marseille-4 specific 134 qPCR, samples are tested using alternative qPCR assays that are specific to other variants that 135 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 8, 2021. ; https://doi.org/10.1101/2021.02.03.21250823 doi: medRxiv preprint circulate at a lower incidence level than the Marseille-4 variant, or they are submitted to next-136 generation sequencing in case of cycle threshold value (Ct) <18 with the SARS-CoV-2 qPCR 137 diagnosis test [1, 11] . Such approach based on qPCR assays targeting specifically SARS-138

CoV-2 variants allows their real-time surveillance, which is warranted to monitor and assess 139 their epidemiological and clinical characterics based on comprehensive sets of data. In The authors have no conflicts of interest to declare. Funding sources had no role in the design 160 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 8, 2021. ; https://doi.org/10.1101/2021.02.03.21250823 doi: medRxiv preprint

Emergence and outcome of the SARS-172

Marseille-4" variant

Emergence and spread of a SARS-CoV-2 175 variant through Europe in the summer of

Early transmissibility 178 assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom

Antigenicity of the SARS-CoV-2 Spike Glycoprotein

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Evidence of SARS-CoV-2 re-197 infection with a different genotype

Reinfection with SARS-CoV-2: Implications for Vaccines

Online ahead of print

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