Recombinase-aided amplification–lateral flow dipstick assay—a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus

The purpose of this study was to explore a specific, simple, and sensitive method for diagnosis of avian infectious laryngotracheitis virus. Recombinase-aided amplification (RAA) and lateral flow dipstick (LFD) were combined for labeling the optimized RAA probe with 6-carboxyfluorescein (FAM) and the 5'-end of the downstream primer with biotin, respectively. By optimizing the reaction time, temperature, and primer concentration of RAA, a RAA-LFD assay, which could be used for detection of infectious laryngotracheitis, was established. After the specificity and sensitivity test, the target gene fragments could be amplified by RAA-LFD assay in 20 min under isothermal conditions (37°C), and the amplification products could be visually observed and determined by LFD within 3 min. There was no cross-reaction with nucleic acids of other avian pathogens, the lowest detectable limit of RAA-LFD was 102 copies/μL, and the sensitivity of this method was 100 times higher than that of conventional PCR with the lowest detectable limit of 104 copies/μL. The results showed that RAA-LFD assay was highly sensitive, easy to use, and more suitable for clinical detection.