A multiplex qRT-PCR assay for detection of Influenza A and H5 subtype targeting new SNPs present in high pathogenicity avian influenza Canadian 2022 outbreak strains

H5N1 is a highly pathogenic avian Influenza A subtype that has been known to also infect mammalian hosts and presents a potential public health risk. To address and mitigate the affects of new SNPs, found in recent Canadian outbreaks, on diagnostic detection we developed two qRT-PCR assays by modifying current probes to match sequences detected in the east and west coast of Canada. These assays were multiplexed with a third qRT-PCR assay targeting the M segment, allowing streamlined detection of Influenza A and subtyping for H5. This three-plex qRT-PCR was validated by assessing analytical specificity, limit-of-detection, precision, and accuracy.