Development of a Rapid Visual Detection Assay for Duck Tembusu Virus Using RT-LAMP-CRISPR/Cas12a

Duck Tembusu virus (DTMUV) is an emerging flavivirus that has inflicted significant economic losses on China's poultry industry. Rapid and accurate detection of DTMUV is crucial for effective prevention and control measures. In this study, we developed a novel, rapid visual detection assay that combines reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) with the CRISPR/Cas12a system for on-site detection of DTMUV. Our results demonstrate that this assay can sensitively and specifically detect the specific DNA plasmids containing the DTMUV <i>NS3</i> gene within 100 min, with a limit of detection as low as 19.3 copies/μL. We successfully applied the RT-LAMP-CRISPR/Cas12a assay to diagnose DTMUV in eight duck embryos and 11 chicken embryonic fibroblast samples, and the results obtained with direct visualization by the naked eye were consistent with those obtained using real-time RT-PCR. Overall, our RT-LAMP-CRISPR/Cas12a assay is a reliable, sensitive, specific, and user-friendly method that holds great promise for early on-site detection of DTMUV in clinical samples, facilitating timely interventions and improved disease management in the poultry industry.