SARS-CoV-2 Evolution and Its Implications for RT-PCR Diagnostic Performance

Mutations in SARS-CoV-2 primer/probe targets can compromise RT-PCR sensitivity, leading to false negatives and undetected viral transmission. This study evaluated mutational landscapes of 20 primer/probe sets targeting E, N, Orf1b-nsp14, RdRp, and S genes using 1,35,852 high-quality genomes across ten countries, five major variants, and three time periods (pre-vaccination, post-vaccination, and recent). Mutations were classified as high-risk or moderate-risk based on their positional susceptibility and mismatch burden. Mutation rate, impact risk, and population frequency within primer/probe targets was calculated along with their geographical-, variant-, and time-dependent variations. Most E, N, RdRp, and S primer/probe targets showed higher and geographically variable mutation rates that increased with time. Variant-wise analysis revealed that mutational accumulation in targets was significantly lower in early variants, that increased sharply with Delta and was most pronounced in Omicron (with its sub-variants). High-risk high-frequency mutations were identified in targets of N-CCDC-forward primer, N-NIH-reverse primer, RdRp-Charité-forward primer, S-Young-forward primer, and S-Sigma (S1/S2)-probes. Moderate-risk high-frequency mutations were observed in E-Charité-forward primer, N-UCDC (N1)-probe, and S-Chan-probe. Mutation frequencies differed across countries due to circulating variants and also across time periods, with some mutations approaching fixation (≥ 95%) in the population. Orf1b-nsp14 and selected N-gene targets like N-Chan, N-NIID, N-UCDC (N2) sets remained mutation-resilient, indicating their diagnostic reliability. A global and regional surveillance-driven, threshold-based strategy which involves periodic mutation monitoring every 6-12 months, consideration of primer/probe redesign when target mutations reach ≥ 10% frequency, and incorporating mutation-resilient targets into multiplex assays is recommended to enhance SARS CoV-2 RT-PCR diagnostic accuracy.