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

Abstract

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.

Keywords: Methodology in biological sciences; Virology.

Graphical abstract

Figure 1

RBM is a key region for viral entry and seroneutralization (A) Primary structure of the SARS-CoV-2 Spike protein. NTD: N-terminal domain; RBD: Receptor-binding domain; RBM: receptor-binding motif; SD1: subdomain 1; SD2: subdomain 2; FP: fusion peptide; HR1: Heptad repeat 1; HR2: Heptad repeat 2; TM: transmembrane region; CT: cytoplasmic region. Amino acid positions of the RBD and RBM boundaries are presented in black and blue numbers, respectively. Functional mutations in the RBM are shown in orange (E484K) and red (N501Y). Purple arrows and numbers correspond to the RT-PCR amplified region. (B) Structure representation of the interaction between SARS-CoV-2 RBD core (green) and hACE2 receptor (white) (PDB 6vw1). RBM is shown in blue. E484 and N501 positions are highlighted in orange and red, respectively. (C) Detection of mutation(s) in a sample compared with a reference sequence. Results of the digestion by a mismatch-specific endonuclease of an auto-control (amplicon of a sample alone) and mix (amplicons of reference and sample) are analyzed by electrophoresis and fragment patterns are detected.

Figure 2

Evaluation of the target amplification and mismatch-specific nuclease assay on reference material (A and B) Linearity of the real-time RT-PCR assay targeting the RBM coding sequence from 10⁵ (blue curve), 10⁴ (red curve), 10³ (green curve), 10² (yellow curve), and 10¹ (pink curve) RNA copy/μL. The linear regression (dashed line) in Panel A has a correlation coefficient R²=0.9986. (C) Endpoint analysis of PCR fragments on gel electrophoresis. (D) Electropherograms of the digestion products. Values above the peaks correspond to the size of the fragment. Peaks on the right (330bp) correspond to the homoduplexes created during the hybridization.

Figure 3

Validation of the assay on SARS-CoV-2 positive clinical samples The First row indicates the number of the samples, from 1 to 8. Ct obtained from TaqPath COVID-19 kit™ (Thermo Fisher) real-time RT-PCR for ORF1ab, S and N genes are presented in the three next rows, respectively (nd: not determined, Ct>35), leading to the following classification: (+) refers to the presence of the deletion indicative of a 501Y.V1 variant, and (-) to a non-501Y.V1 sample. Below the table, the results of the mismatch-specific assay followed by capillary electrophoresis are presented in a “gel-like” format for the eight samples. The molecular weight ladder is indicated on the left. For each sample (1 to 8), the experiment was conducted in three conditions: The first lane is the self-hybridization (sample/sample; Lane A), the second one is sample/BavPat1 reference (lane B), and the third sample/501Y.V1 reference (Lane C). The fragment above 330 bp corresponds to uncleaved homoduplexes.