2015年以来,我国爆发了一起由禽腺病毒血清型4(FAdV-4)引起的肉鸡传染病,造成了巨大的经济损失。快速,准确,特异性检测对FAdV-4的预防和控制具有重要意义。在这项研究中,建立了一种结合环介导等温扩增(LAMP)和热球菌(PfAgo)的FAdV-4检测方法。特异性引物,指导DNA(gDNA)和分子信标被设计为靶向FAdV-4六邻体基因的保守区域。优化反应条件后,该测定的最低检测量可以达到5个拷贝。它仅扩增FAdV-4,并且不存在与其他病原体的交叉反应性。该测定仅花费约50分钟,结果可以在紫外线或蓝光下用肉眼看到,摆脱专门的仪器。通过使用20个临床样品验证了这种新型LAMP-PfAgo测定,结果与金标准实时聚合酶链反应方法相同。总之,本文建立的LAMP-PfAgo测定法提供了一种快速、可靠,方便,用于FAdV-4的现场检测和临床诊断的超灵敏和高度特异性工具。
Since 2015, an outbreak of an infectious disease in broilers caused by fowl adenovirus serotype 4 (FAdV-4) has occurred in China, resulting in substantial economic losses. Rapid, accurate, and specific detection are significant in the prevention and control of FAdV-4. In this study, an FAdV-4 detection method combining loop-mediated isothermal amplification (LAMP) and Pyrococcus furiosus Argonaute (PfAgo) was established. Specific primers, guide DNAs (gDNAs), and molecular beacons were designed to target a conserved region of the FAdV-4 hexon gene. After optimizing the reaction conditions, the minimum detection of this assay could reach 5 copies. It only amplified FAdV-4, and there was no cross-reactivity with other pathogens. The assay took about only 50 min, and the results could be visualized with the naked eye under ultraviolet or blue light, getting rid of specialized instruments. This novel LAMP-PfAgo assay was validated by using 20 clinical samples and the results were identical to gold-standard real-time polymerase chain reaction method. In summary, the LAMP-PfAgo assay established in the paper provides a rapid, reliable, convenient, ultra-sensitive and highly specific tool for the on-site detection and clinical diagnosis of FAdV-4.