Abstract / Summary
Abstract Background: Real-time RT-PCR assays targeting the influenza A matrix (M) gene are widely used in clinical laboratories for routine detection of H5N1 infections. Viral genetic drift may compromise primer and probe binding efficiency, potentially affecting assay sensitivity. Continuous evaluation of binding site stability is therefore essential for diagnostic reliability.
Methods: Forty-nine full-length matrix gene sequences of influenza A (H5N1) collected between 1996–2025 and included isolates from: Homo sapiens, Avian species, other mammalian hosts (accession numbers provided on the tables). Only sequences covering nucleotide positions 79–175 were included. Gene sequences were retrieved from the NCBI GenBank database. Multiple sequence alignment was performed using (MUSCLE in MEGA software). Primer and probe binding regions corresponding to coordinates 79–97 (forward primer), 120–138 (probe), and 154–175 (reverse primer) were evaluated for nucleotide mismatches. Frequencies of substitutions were calculated and stratified by host and temporal distribution.
Results: All sequences fully covered the evaluated binding regions. The majority of isolates demonstrated complete conservation within primer and probe sites. Observed substitutions were infrequent and primarily single nucleotide mismatches. No extensive multi-site disruptions were identified within any binding region. Substitution patterns showed temporal clustering but did not suggest widespread destabilization of assay target regions.
Conclusion: The results indicate that the evaluated matrix gene RT-qPCR assay demonstrates substantial tolerance to isolated nucleotide substitutions, with most circulating strains remaining within predicted tolerance limits. Observed limited mismatches are unlikely to significantly compromise diagnostic assay performance. Continued molecular surveillance of primer and probe binding sites is recommended to maintain analytical sensitivity in clinical laboratories.