Endonuclease-based genotyping of the RBM as a method to track the emergence or evolution of SARS-CoV-2 variants

Eva Lopez (1) , Margot Barthélémy (1) , Cécile Baronti (1) , Shirley Masse (2) , Alessandra Falchi (2) , Fabien Durbesson (3) , Renaud Vincentelli (3) , Xavier de Lamballerie (1) , Rémi Charrel (1, 4) , Bruno Coutard (1)

1 UVE - Unité des Virus Emergents
2 Università di Corsica Pasquale Paoli [Université de Corse Pascal Paoli]
3 AFMB - Architecture et fonction des macromolécules biologiques
4 APHM - Assistance Publique - Hôpitaux de Marseille

Since the beginning of the COVID-19 pandemics, variants have emerged. Some of them display increased transmissibility and/or resistance to immune response. Most of the mutations involved in the functional adaptation are found in the receptor-binding motif (RBM), close to the interface with the receptor ACE2. We thus developed a fast molecular assay to detect mutations in the RBM coding sequence. After amplification, the amplicon is heat-denatured and hybridized with an amplicon of reference. The presence of a mutation can be detected using a mismatch-specific endonuclease and the cleavage pattern is analyzed by capillary electrophoresis. The method was validated on RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants produced in vitro before being implemented for clinical samples. The assay showed 97.8% sensitivity and 97.8% specificity. The procedure can be set up for high-throughput identification of the presence of mutations and serve as a first-line screening to select the samples for full genome sequencing.

Mots clés: Methodology in biological sciences; Virology

Domaines: Sciences du Vivant [q-bio]

Eva Lopez, Margot Barthélémy, Cécile Baronti, Shirley Masse, Alessandra Falchi, et al.. Endonuclease-based genotyping of the RBM as a method to track the emergence or evolution of SARS-CoV-2 variants. iScience, 2021, 24 (11), pp.103329. ⟨10.1016/j.isci.2021.103329⟩. ⟨hal-03524036⟩