Chen Jimin, Tao Dagang, Yang Fan, Pan Chengfu, Bao Xinguo, Xie Shengsong, Gong Ping, Zhao Changzhi, Lin Ruiyi
College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China.
Animals (Basel). 2024 Nov 27;14(23):3439. doi: 10.3390/ani14233439.
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 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.
鸭坦布苏病毒（DTMUV）是一种新出现的黄病毒，给中国家禽业造成了重大经济损失。快速准确地检测DTMUV对于采取有效的预防和控制措施至关重要。在本研究中，我们开发了一种新型的快速可视化检测方法，该方法将逆转录环介导等温扩增（RT-LAMP）与CRISPR/Cas12a系统相结合，用于DTMUV的现场检测。我们的结果表明，该检测方法能够在100分钟内灵敏且特异地检测到含有DTMUV基因的特定DNA质粒，检测限低至19.3拷贝/μL。我们成功地将RT-LAMP-CRISPR/Cas12a检测方法应用于诊断8个鸭胚和11个鸡胚成纤维细胞样本中的DTMUV，肉眼直接观察获得的结果与实时RT-PCR获得的结果一致。总体而言，我们的RT-LAMP-CRISPR/Cas12a检测方法是一种可靠、灵敏、特异且用户友好的方法，在临床样本中对DTMUV进行早期现场检测具有很大的前景，有助于家禽业及时进行干预并改善疾病管理。
