Avian influenza virus (AIV) infection can lead to severe economic losses in the poultry industry and causes a serious risk for humans. A rapid and simple test for suspected viral infection cases is crucial. In this study, a reverse transcription recombinase-aided amplification assay (RT-RAA) for the rapid detection of all AIV subtypes was developed. The reaction temperature of the assays is at 39 °C and the detection process can be completed in less than 20 min. The specificity results of the assay showed that this method had no cross-reaction with other main respiratory viruses that affect birds, including Newcastle disease virus (NDV) and infectious bronchitis virus (IBV). The analytical sensitivity at a 95% confidence interval was 10 RNA copies per reaction. In comparison with a published assay for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), the κ value of the RT-RAA assay in 384 avian clinical samples was 0.942 (p < 0.001). The sensitivity and specificity of the RT-RAA assay for avian clinical sample detection was determined as 97.59% (95% CI 93.55-99.23%) and 96.79% (95% CI 93.22-98.59%), respectively. The RT-RAA assay for AIV in this study provided an effective and practicable tool for AIV molecular detection.
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