Reverse transcription recombinase-aided amplification assay combined with a lateral flow dipstick for detection of...

Duck Tembusu virus disease, caused by duck Tembusu virus (DTMUV), brings great harm to duck industry. Early diagnosis is of great significance for the prevention and control of this disease. In order to develop a specific and sensitive method for rapid diagnosis of DTMUV, reverse-transcriptase recombinase aided amplification combined with lateral flow dipstick (RT-RAA-LFD) method for detection of DTMUV was established. Firstly, downstream primer was labeled with biotin and probe was labeled with FAM, and primer concentration, reaction time, and reaction temperature were optimized. Then, the specificity and sensitivity of this method was investigated. The results of specificity test showed that it had no cross reaction with other common pathogens such as low pathogenic avian influenza virus (AIV), Newcastle disease virus (NDV), duck hepatitis A virus (DHV), and duck Reovirus. The results of sensitivity test showed that the minimum detection limit of this method was 10 copies/μL, which was 1000 times than conventional RT-PCR (10⁴ copies/μL), and equivalent to that of fluorescent quantitative PCR. Furthermore, this RT-RAA-LFD method demonstrated excellent intragroup and intergroup consistency. Finally, the RT-RAA-LFD assay and real-time PCR were both utilized to examine 58 clinical samples concurrently. The results showed that the RT-RAA-LFD method (5/58) was more sensitive than the fluorescence quantitative PCR method (4/58). In summary, RT-RAA-LFD method established in this study had a strong specificity and high sensitivity, which provided technical support for clinical detection of DTMUV.