Identificação in silico de mutações das variantes de preocupação do SARS-CoV-2 em sítios de anelamento e redesenho dos primers e sondas de RT-qPCR afetados

COVID-19, caused by SARS-CoV-2, exhibits high genetic variability, and mutations in regions recognized by primers and probes may compromise the reliability of RT-qPCR tests. This study assessed the impact of variant-of-concern mutations on the annealing sites of diagnostic assays and proposed corrected sets for the affected assays. This is a descriptive-analytical in silico study. Six complete genomes, the Wuhan-Hu-1 reference and one representative of each variant of concern (Alpha, Beta, Gamma, Delta and Omicron), were retrieved from GenBank and aligned with Clustal Omega. Forty oligonucleotides from five protocols (CDC, Charité/WHO, China CDC, HKU and Institut Pasteur) were mapped onto the reference genome. After consolidating sequences identical across protocols, 37 unique oligonucleotides organised into 12 sets were analysed. Thirty remained intact across the five variants and seven displayed at least one mismatch, totalling 18 events, all non-synonymous, distributed over 11 genomic positions. The China CDC N set concentrated 12 of these events by overlapping the 28,881–28,883 cluster, the origin of the R203K and G204R substitutions. One event was classified as highly critical: the substitution at position 15,451 (ORF1b:G662S, Delta variant), located one base from the 3′ end of the forward primer of the RdRP_SARSr set. New sets were designed for the five affected assays, restricted to the 28,825 positions (96.4% of the genome) conserved across all six sequences, showing complete complementarity and adequate thermodynamic parameters. Specificity screening against the six reference human coronaviruses indicated no expected reactivity with MERS-CoV or with the seasonal coronaviruses, although the sets targeting ORF1b and the E gene retain recognition of SARS-CoV, a property inherited from the original pan-Sarbecovirus design. Global genome conservation does not ensure the integrity of diagnostic sites, and monitoring these regions should be integrated into routine genomic surveillance.