Reverse transcription recombinase-aided amplification assay combined with a lateral flow dipstick for detection of avian infectious bronchitis virus

Abstract

The study was conducted to develop a specific, simple, and sensitive method for diagnosis of avian infectious bronchitis virus (IBV). In this experiment, the selected downstream primer was labeled with biotin and the 5′ end of RAA probe was labeled with FAM by reverse transcription recombinase-aided amplification (RT-RAA) combined with lateral flow dipstick (LFD). A RT-RAA-LFD assay that could be used for detection of IBV was established after optimization of RT-RAA reaction time, reaction temperature, and primer concentration. This method did not need reverse transcription of IBV template under isothermal condition (37°C), the amplification of target gene fragments could be completed within only 24 min, and the amplification products could be visually observed and determined by LFD within 3 min. The specificity test demonstrated that there was no cross reaction with the nucleic acids of other similar common pathogens. The lowest detectable limit for IBV was 102 copies/μL, and this method was 100 times more sensitive than conventional PCR (104 copies/μL), as verified by sensitivity test. The results showed that RT-RAA-LFD assay with strong specificity and high sensitivity was simple and easy to operate, and could be used for rapid detection of IBV in clinical diagnosis.