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

Abstract

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.