Development and application of a real-time fluorescent RAA assay for H9 subtype avian influenza viruses

The H9N2 subtype of avian influenza virus (AIV) is a critical pathogen responsible for avian infectious diseases, inducing respiratory symptoms in poultry and exhibiting high susceptibility to coinfections, which complicates clinical diagnosis. In this study, we designed specific primers and probes targeting the hemagglutinin (HA) gene of H9N2 AIV and developed a real-time fluorescent reverse transcription recombinase-aided isothermal amplification (RT-RAA) assay through systematic optimization of reaction components and conditions. The established method demonstrated exclusive reactivity with the H9N2 subtype, yielding negative results for all other tested viral pathogens, thereby showcasing high specificity. Analytical sensitivity testing revealed the capability to detect as low as 13.5 copies/μL of H9N2 viral RNA, indicating superior sensitivity. To further validate the practical application of this method, a total of 48 clinical samples, comprising oropharyngeal and cloacal swabs, were tested using the developed RT-RAA assay. The results revealed a positivity rate of 79%, reflecting strong diagnostic performance. This study presents a rapid RT-RAA assay characterized by high specificity and sensitivity, offering a robust technical platform for the immediate identification of H9N2 AIV and facilitating epidemiological surveillance in avian populations.