Identification of Major SARS CoV-2 Variants in India using in silico PCR-RFLP analysis

PURPOSE: As of June 30, 2021, WHO reported 181,344,224 confirmed cases worldwide and 3,934,252 deaths due to SARS CoV-2. India accounts almost 10% of the total mortality. The mutated Variants of Concern (VoC) and Variants of Interest (VoI) has acquired non-synonymous amino acid substitutions in Spike (S), ORF1a, ORF1b, Nucleocapsid (N), Membrane (M), Envelope (E), ORF6, ORF7a, ORF3a and ORF8 regions exhibiting more virulence and higher transmission rate. The S gene displaying nucleotide polymorphisms are studied for identification of major SARS CoV-2 variants circulating in India using in silico PCR-RFLP analysis. METHODS & MATERIALS: DNA sequences of major SARS CoV-2 variants [alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), epsilon (B.1.429+B.1.427), eta (B.1.525) and zeta (P.2)] prevailing in India retrieved from GISAID database were annotated using VIPR-VIGOR4 genome annotator tool. The S gene of variants were aligned using CLUSTALW algorithm in MEGA-X with default parameters and analyzed for sequence identity using Geneious Prime v2019.2.1. Primers were designed using Primer-BLAST tool, proof-read using FastPCR v6.7.46 and PCR-amplified using Snapgene v.3.2.1. Unique restriction sites in S amplicons of each variant were subjected to online Restriction Analyzer tool. The S amplicons were digested with restriction endonucleases and the band profile of each variant were visualized in the gel simulation tool using Snapgene v.3.2.1. RESULTS: The percentage of identical sites present in S region among SARS CoV-2 variants was found to be 98.6%. The amplified products were in the length of 3,689 and 3,698 bp. Out of 400 restriction endonucleases identified, 14 buffer-compatible enzymes were selected for single-step restriction digestion to generate unique RFLP profiles for each variant. The BsaXI-XcmI-AcuI triple digest showed unique banding pattern identifiers on 2% agarose gel for individual variants including alpha, eta, delta and zeta. The ApaLI-BsaI double digest produced distinct band profiles for beta and epsilon variants. CONCLUSION: Our study strongly suggests PCR-RFLP analysis of Spike region can differentiate major SARS CoV-2 variants that are circulating in India. Further, quadruple digestion based wet lab experiments are underway to explore the possibility of surveillance of the major variants using a single-tube reaction followed by agarose gel-based profiling.