Development of multiplex real-time RT‒PCR assays for the simultaneous detection and lineage differentiation of H9 avian influenza viruses.

H9 avian influenza viruses (AIVs) have caused continuous economic losses in poultry and pose a risk of introducing novel lineages through wild birds and human activities, highlighting the importance of rapid detection and lineage discrimination for effective surveillance. In this study, we developed a multiplex real-time reverse transcription PCR (rRT‒PCR) assay that simultaneously detects the H9 gene and differentiates the Y and B lineages using a single set of common primers with one H9 common probe (H9-Probe) and two B and Y lineage-specific probes (B-Probe and Y-Probe). The assay displayed high analytical specificity (100%) in detecting H9 and differentiating B and Y lineages in tests with 50 reference strains, 6 synthetic G lineage H9 genes and 28 field samples. Experiments with synthesized G lineage constructs showed fluorescence signals with the H9-Probe but not the B-Probe and Y-Probe, suggesting its potential to indicate the presence of G lineage AIVs. The limit of detection was 10-10² EID₅₀, and the performance with field samples was comparable to that of a commercial kit. In the mixed B and Y lineage sample, each lineage was reliably detected and distinguished according to its relative concentration, but detection was slightly delayed when the sample was mixed with high concentrations of the virus. Collectively, this multiplex H9 rRT‒PCR assay provides a sensitive, specific, and cost-efficient tool for the simultaneous detection and lineage differentiation of H9 AIVs, supporting the large-scale surveillance and early identification of emerging variants.