Establishment and performance evaluation of quadruplex RT-qPCR method for universal avian influenza virus detection and H5/H7/H9 subtyping

Avian influenza viruses (AIVs) are zoonotic pathogens that exhibit periodic pandemics globally, posing significant public health risks. In this study, we developed a quadruplex RT-qPCR method that targets the M gene for universal AIV detection and the HA gene for simultaneous differentiation of the H5, H7, and H9 subtypes. The assay achieved a detection limit of 1 copy/μL for the H5, H7, and H9 reference materials based on HA gene quantification, equivalent to 5 copies per reaction. No cross-reactivity was observed with a panel of common avian pathogens. The reproducibility evaluation demonstrated both intra-batch and inter-batch coefficients of variation (CVs) to be less than 5%. Furthermore, the assay results were consistent with the expected outcomes for the reference materials tested, and the subtyping results were in agreement with whole-genome sequencing for the environmental samples. These findings indicate that the quadruplex RT-qPCR assay is a reliable tool for broad AIV detection and H5/H7/H9 subtyping, supporting its application in AIV surveillance.