鸭腺病毒3型LAMP快速检测方法的建立

Establishment of LAMP method for rapid detection of duck adenovirus 3

鸭腺病毒3型(DAdV-3)是我国鸭群中出现的一种新型腺病毒,属于腺病毒科禽腺病毒属成员。为建立DAdV-3的快速检测方法,本研究根据DAdV-3的fiber2基因,设计特异性的LAMP检测引物,构建重组质粒标准品pT-DAdV-3,经各反应条件优化,建立了DAdV-3 LAMP快速检测方法。结果显示,建立的LAMP检测方法最佳反应条件为64℃40 min;该方法仅对DAdV-3呈阳性反应,与番鸭细小病毒、鸭圆环病毒、鸭源减蛋综合征病毒等其他常见鸭源传染病病原均无交叉反应,特异性强。该方法对重组质粒标准品的最低检测限为50拷贝/μL;利用本研究建立的LAMP方法、前期建立的PCR方法和基于MGB探针的荧光定量PCR方法对临床收集的66份鸭组织病料样品进行检测,结果显示阳性率分别为13.63%(9/66)、13.63%(9/66)和15.15%(10/66);且LAMP检测的阳性样品经PCR方法检测和荧光定量PCR方法检测均为阳性,符合率为100%。本研究首次建立了检测DAdV-3的LAMP方法,该方法快速敏感、特异性强,为该病的流行病学调查提供技术支撑。

Duck adenovirus 3(DAdV-3),a newly identified adenovirus,which belongs to Aviadenovirus under Adenoviridae,was recently discovered in duck flocks in China.In order to establish a rapid detection method for DAdV-3,a set of specific LAMP method primers were designed based on the characteristics of fiber2 gene of DAdV-3,and then a rapid LAMP method for DAdV-3was established by optimizing the reaction conditions.The results demonstrated that the optimal reaction condition of LAMP method were 64℃40 minutes,with the minimum detection limit was 50 copies/μL.The method showed good specificity,which only amplified DAdV-3 with no cross-reactivity observed by using other duck origin viruses(such as Muscovy duck parvovirus,duck circovirus and duck origin egg drop syndrome virus and so on).A total of 66 clinically collected duck tissue samples were tested by the optimized LAMP method,the previously reported PCR and the MGB-based real-time fluorescence quantitative PCR(qPCR)methods,and the positive rates were 13.63%(9/66),13.63%(9/66)and 15.15%(10/66),respectively.All the positive samples detected by LAMP were also tested positive by PCR and qPCR methods,and the coincidence rate was 100%.In this study,a LAMP method for detecting DAdV-3 was established for the first time,which is rapid,sensitive and specific,and provides technical support for the epidemiological investigation of the disease in the field.