Quadruplex real-time RT-qPCR for simultaneous detection and subtyping of avian influenza viruses and avian coronaviruses.

Avian influenza viruses (AIVs) and avian coronaviruses (ACoVs) are major avian pathogens that can spread via migratory birds, posing significant threats to poultry and public health. Highly pathogenic AIVs (HPAIVs) are primarily associated with the H5 subtype, whereas avian coronaviruses mainly belong to the Gamma- and Delta- genera. However, current molecular diagnostic methods are largely limited to single-target detection, hindering efficient surveillance of multiple pathogens and high-risk subtypes. In this study, we developed a quadruplex TaqMan probe-based RT-qPCR assay that enables simultaneous detection of AIV (M gene), identification of the H5 subtype (HA gene), and differentiation of avian Gamma- and Delta-coronaviruses (RdRp gene) within a single reaction. The assay exhibited high sensitivity, with limits of detection of 1 copy/μL for AIV-M, H5 (M+HA), ACoV-Gamma, and 10 copies/μL for ACoV-Delta. No cross-reactivity was observed among 26 common pathogens, and intra-assay variability was low (CV < 2.83%), indicating high specificity and reproducibility. Validation using 70 clinical samples showed over 98.57% concordance with Sanger sequencing, accurately identifying both single and mixed infections. Overall, this assay provides a sensitive, specific, and cost-effective platform for the simultaneous detection and surveillance of multiple avian viruses. By enabling rapid multiplex detection of co-circulating pathogens, it may facilitate early warning and surveillance of viral diseases in poultry and wild birds and support the early identification and control of potential zoonotic threats.