Five years of SARS CoV-2 Evolution: Implications for RT PCR Diagnostic Performance

Mutations in SARS-CoV-2 primer/probe target regions can compromise RT-PCR sensitivity, leading to false negatives and undetected viral transmission. This study evaluated mutational landscapes of 20 different primer/probe sets targeting E , N , Orf1b-nsp14 , RdRp , and S genes, using 86,250 high-quality genomes from seven countries. A time-dependent mutational analysis was also conducted through pre-vaccination, post-vaccination, and recent periods. Mutations were classified as high-risk (causing assay failure) or moderate-risk (reducing assay sensitivity). Overall mutation rates, mutational risk, specific mutation frequencies, along with their geographical and time-dependent variations were examined. E , N , RdRp , and S target regions showed significantly higher and geographically variable mutation rates (e.g., RdRp ’s Charité Germany-forward primer; S -targeted Young Singapore-forward primer), that rose sharply post-vaccination. High-risk high-frequency mutations included G28881A/G28882A/G28883C ( N , CDC China-forward primer), 9-nt deletion ( N , NIH Thailand-reverse primer), G15451A ( RdRp , Charité Germany-forward primer), 6-nt deletion ( S , Young Singapore-forward primer), and T23599G/C23604A ( S , Sigma Aldrich probes). Moderate-risk high-frequency mutations involved C26270T ( E , Charité Germany-forward primer), C28311T ( N , CDC USA-N1 probe), and G22813T ( S , Chan China-probe). Specific mutation frequencies also varied geographically, with some approaching fixation over time. HKU’s Orf1b-nsp14 and N (Chan China, NIID Japan, CDC USA-N2) sets remained mutation-resilient. Based on mutation emergence and fixation timelines, primers and probes are recommended to be re-evaluated/ re-designed every 6-12 months. Target regions with high-risk high-frequency mutations require immediate replacement, while those with moderate risk high-frequency mutations need regular surveillance. Cumulatively acting low-frequency mutations also require monitoring. Incorporating mutation-resilient target sets into multiplex assay designs can help preserve diagnostic accuracy.