Importance: Avian influenza virus (AIV) is classified into subtypes by hemagglutinin (HA) and neuraminidase (NA) genes. Wild waterfowl harbor H1-H16 and N1-N9 subtypes. Large-scale AIV surveillance requires substantial labor, time, and cost when using conventional HA and NA subtyping methods.

Objective: This study aimed to develop multiplex reverse transcription polymerase chain reaction (RT-PCR) and universal assays to detect all H1-H15 and N1-N9 subtypes in just eight RT-PCR reactions.

Methods: Subtype-specific forward primers and conserved reverse primers were used to identify HA and NA subtypes based on RT-PCR amplicon size. The HA cleavage site was characterized by sequencing. For NA, a universal forward primer enabled sequencing-based subtyping from a single reaction with the universal reverse primer.

Results: Seventy reference strains and 113 low pathogenic AIVs (from 2021-2022 and 2022-2023 season), covering H1-H15 and N1-N9 subtypes, were tested. The performance of this method was nearly complete, with a few subtype/cleavage site exceptions in relatively long-stored samples. The assays showed 98.90% sensitivity for HA (181/183), 98.91% for NA (182/184), and 100% specificity for both of HA and NA subtyping.

Conclusions and relevance: These subtyping assays offer a rapid, sensitive, and cost-effective method for AIV subtyping, enhancing efficiency in large-scale surveillance.