Influenza A virus has eight negative-sense single-stranded RNA genome segments and is classified according to the subtype of two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Avian influenza virus (AIV) is an Influenza A virus with 16 HA subtypes and 9 NA subtypes; wild waterfowl can be infected with all AIV subtypes, and those viruses are transmissible to domestic birds (Krammer et al., 2018). AIVs can cause mild to severe disease and can be classified as a low-pathogenicity avian influenza viruses (LPAIVs) or a high-pathogenicity avian influenza viruses (HPAIVs) according to their virulence in poultry (WOAH, 2023). All the HPAIVs identified belong to the H5 or H7 subtype, and H5 HPAIVs of the Gs/GD lineage have been successful at propagating worldwide and evolved into many different sublineages through genomic rearrangements during circulation in wild waterfowl and poultry (G.C.f.H.N.R.I, 2016, Verhagen et al., 2015). Among them, clade 2.3.4.4 H5 HPAIVs have been prevalent in many regions in the world since 2014, and recently, clade 2.3.4.4b H5N1 HPAIV emerged in Europe in 2020 and has continued to cause unprecedented economic losses in the poultry industry and high mortality in wild birds worldwide (Lee et al., 2014, Shi et al., 2023a). H7 HPAIV outbreaks are much less common than H5 HPAIV outbreaks, but there is potential for these viruses to cause the next pandemic via the acquisition of human-to-human transmissibility (Cowling et al., 2013, infections, 2023b). H9N2 LPAIVs are generally divided into three lineages, Y439, Y280, and G1, and the Y280 and G1 lineages are endemic to poultry in many countries, especially in the live poultry market (LPM), with rare cases of human infection (Peacock et al., 2019, Sagong et al., 2023b). Although LPAIV does not cause high mortality in poultry in general, it should be controlled because H5 and H7 HPAIVs can arise during the circulation of H5 and H7 LPAIV in poultry, and H9 LPAIV may cause considerable economic losses through mortality and decreased egg production in layers (Alexander and Brown, 2009).

For accurate and rapid detection of AIV, molecular diagnostic methods such as RT-PCR and real-time RT-PCR (rRT-PCR) are the preferred options because they can quickly detect the presence of AIV and its subtypes in inactivated viral samples without requiring high-biosafety-level facilities to amplify infectious viruses from eggs or cells (WOAH, 2023). Among the many molecular diagnostic methods, fluorescence-based rRT-PCR has the capacity to detect smaller amounts of viral genes than RT-PCR and can quantify the number of viral genes from the number of threshold cycles based on a standard curve (Kralik and Ricchi, 2017). The diagnostic ability of rRT-PCR has been rapidly improved to achieve high sensitivity and specificity via the development of new technologies, such as internal positive controls, TaqMan minor groove binding (MGB) probes, and one-step rRT-PCR components with high PCR performance (Di Trani et al., 2006, Schweiger et al., 2000, Spackman et al., 2002, Suarez et al., 2007). Many rRT-PCR methods have been designed for the detection and subtyping of AIVs, and most of them target H5, H7 or H9 genes, the most problematic subtypes in the poultry industry (Liu et al., 2018, Monne et al., 2008, Panzarin et al., 2022, Suarez et al., 2007, Yang et al., 2022, Zhang et al., 2017), and some of them were designed to be performed in single tube using sets of primers and probe specific to each subtype labeled with a unique fluorescent dye (Monne et al., 2008, Yang et al., 2022, Zhang et al., 2017). However, those subtype-specific rRT-PCRs, in a singleplex or multiplex form, often fall short of complete coverage of all the lineages in a subtype because they tend to focus on the specific genetic groups circulating locally.

In Korea, given the continual H5 HPAIV epidemic, the Animal and Plant Quarantine Agency (APQA) developed a singleplex rRT-PCR for the Matrix, H5, and H7 genes in 2017 and has been using them successfully to carry out large-scale national surveillance in collaboration with many local veterinary service laboratories in each province (An et al., 2023, Heo et al., 2021, Kim et al., 2018, Lee et al., 2014, Lee et al., 2007, Lee et al., 2018, Sagong et al., 2022). Additionally, the H9-specific singleplex rRT-PCR has also been developed and validated recently (Sagong et al., 2023a). However, as the number of tests for national AI active surveillance has increased greatly in Korea due to the HPAI outbreaks and reinforced surveillance, the workload for subtyping on the Matrix gene-positive cases increased proportionally nationwide. In this study, to perform those detecting and subtyping tests with speed and efficiency, multiplex rRT-PCR method for the H5, H7 and H9 genes with three sets of primers and probe, without cross-reactivity and with a wider coverage in terms of genomic variations, was developed and compared with in-house singleplex rRT-PCR methods.