Evaluating primer and probe mismatch tolerance in an Influenza A matrix gene RT-qPCR using contemporary human and zoonotic strains

Abstract Background Genetic drift and host-associated adaptation in influenza A viruses threaten the long-term reliability of RT-qPCR–based diagnostics, particularly when nucleotide mismatches arise within primer and probe binding regions. Conventional assay evaluations often emphasize sequence conservation but rarely assess functional mismatch tolerance across divergent subtypes and hosts. Methods We performed an in silico evaluation of a matrix (M) gene–targeted RT-qPCR assay by aligning primer and probe binding regions against 22 H1N1 isolates and representative H3N2 and H5N1 reference strains, including recent zoonotic isolates from avian and bovine hosts. Nucleotide mismatches were identified, quantified, and mapped relative to assay components and oligonucleotide termini. Mismatch burden was summarized by subtype and assay region. Results H1N1 isolates exhibited complete conservation across primer and probe regions. In contrast, H3N2 and H5N1 strains demonstrated subtype-specific sequence variability, with a total of eleven mismatches identified across seven non-H1N1 isolates (mean mismatch per isolate = 2.43). Probe mismatches predominated (63.6%), occurring primarily at internal positions, while primer mismatches were infrequent and largely avoided 3′ terminal nucleotides. Recent H5N1 isolates (2023–2024) shared conserved internal mismatches in the probe and forward primer, whereas a historical H5N1 isolate (2016) exhibited a distinct profile including a terminal probe mismatch. Despite this variability, mismatch patterns were consistent with preserved amplification potential. Conclusion This study demonstrates that the evaluated influenza A M gene RT-qPCR assay exhibits inherent mismatch tolerance across human and zoonotic subtypes. By shifting diagnostic evaluation from strict sequence identity to functional resilience, our findings provide a framework for designing and maintaining robust molecular assays suitable for surveillance and pandemic preparedness amid ongoing viral evolution. Graphical Abstract In silico evaluation of an influenza A matrix gene RT-qPCR assay demonstrates subtype-specific primer and probe mismatches across H3N2 and H5N1 strains, including recent zoonotic isolates. Despite observed variability, mismatches predominantly occur at internal positions and spare primer 3′ termini, supporting inherent assay mismatch tolerance and suitability for surveillance applications.