Establishment of a Real-Time Fluorescence Isothermal Recombinase-Aided Amplification Method for the Detection of H9 Avian Influenza Virus.

The H9 subtype of avian influenza virus (AIV) has been characterized by its rapid spread, wide range of prevalence, and continuous evolution in recent years, leading to an increasing ability for cross-species transmission. This not only severely impacts the economic benefits of the aquaculture industry, but also poses a significant threat to human health. Therefore, developing a rapid and sensitive detection method is crucial for the timely diagnosis and prevention of H9 AIVs. In this study, a real-time fluorescent reverse transcription recombinase-aided isothermal amplification (RT-RAA) technique targeting the hemagglutinin (HA) of H9 AIVs was established. This technique can be used for detection in just 30 min at a constant temperature of 42 °C, and it exhibits good specificity without cross-reactivity with other viruses. Sensitivity tests revealed that the detection limit of RT-RAA was 163 copies per reaction, and the visual detection limit was 1759 copies per reaction at a 95% confidence interval, both of which are capable of detecting low concentrations of standards. Furthermore, RT-RAA was applied to detect 155 clinical samples, and compared to real-time fluorescent quantitative PCR (RT-qPCR), RT-RAA demonstrated high accuracy, with a specificity of 100% and a kappa value of 0.96, indicating good correlation. Additionally, with the assistance of a portable blue imaging device, we can visually observe the amplification products, greatly facilitating rapid detection in resource-limited environments. The RT-RAA detection method developed in this study does not require expensive equipment or highly skilled staff, making it beneficial for the accurate and low-cost detection of H9 AIVs.