Background: The H5 subtype avian influenza virus (AIV) has caused huge economic losses to the poultry industry and is a threat to human health. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks.

Methods: In this study, we developed a reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of H5 subtype AIV. Assays were performed at a single temperature (39 °C), and the results were obtained within 20 min.

Results: The assay showed no cross-detection with Newcastle disease virus or infectious bronchitis virus. The analytical sensitivity was 103 RNA copies/μL at a 95% confidence interval according to probit regression analysis, with 100% specificity. Compared with published reverse transcription quantitative real-time polymerase chain reaction assays, the κ value of the RT-RAA assay in 420 avian clinical samples was 0.983 (p < 0.001). The sensitivity for avian clinical sample detection was 97.26% (95% CI, 89.56-99.52%), and the specificity was 100% (95% CI, 98.64-100%).

Conclusions: These results indicated that our RT-RAA assay may be a valuable tool for detecting H5 subtype AIV.