鸭腺病毒A型TaqMan实时荧光定量PCR检测方法的建立

试验旨在建立鸭腺病毒A型（duck adenovirus A，DAdV-A）TaqMan实时荧光定量PCR检测方法。根据DAdV-A Hexon基因序列设计特异性引物和探针，建立了基于TaqMan探针检测DAdV-A的实时荧光定量PCR检测方法，对其特异性、灵敏性、重复性进行检测，用建立的TaqMan实时荧光定量PCR检测方法和常规PCR方法同时对福建地区临床收集的85份番鸭源病料进行DAdV-A感染的检测，比较其符合率。结果表明，试验成功建立了检测DAdV-A的实时荧光定量PCR检测方法，其扩增相关系数为0.996，扩增效率为99.9%；特异性强，对鸭常见病原（如鸭瘟病毒、鹅细小病毒、番鸭细小病毒、鸭圆环病毒、鸭源大肠杆菌、鸭疫里默氏杆菌和鸭源禽多杀性巴氏杆菌）检测均为阴性；灵敏度高，最低检测限为8.37拷贝/μL；重复性好，组内变异系数和组间变异系数分别为0.54%~1.28%和0.61%~2.39%。对临床送检的85份病料，TaqMan实时荧光定量PCR方法的阳性率为7.06%（6/85），PCR方法的阳性率为5.88%（5/85），且PCR检测的阳性样品经TaqMan实时荧光定量PCR方法检测均为阳性，符合率为100%。本研究建立了基于TaqMan探针检测DAdV-A的实时荧光定量PCR检测方法，为鸭群中开展DAdV-A的分子流行病学研究提供了有效技术手段。

Establishment of a TaqMan-based Real-time PCR Method of Duck Adenovirus A

This study was aimed to establish TaqMan-based Real-time PCR method of duck adenovirus A (DAdV-A).According to the sequence of DAdV-A Hexon gene,the specific primers and probe were designed to develop the TaqMan-based Real-time PCR method,and the specificity,sensitivity and repeatability of the established method were detected.85 clinic samples were tested by the established TaqMan-based Real-time PCR method,then the coincidence rate were calculated which compared with the conventional PCR.The results showed that a TaqMan-based Real-time PCR method was successfully established,the correlation (R²) was 0.996,and the efficiency was 99.9%.This method had strong specificity,high sensitivity and good repeatability.No amplification was detected from common duck origin pathogens,such as,duck virus enteritis,goose parvovirus,Muscovy duck virus,duck circovirus,Escherichia coli,Rimerella anatipstifer and Pasteurella multocida.The limit of detection concentration was 8.37 copies/μL.The intra-and inter-assay were ranged from 0.54% to 1.28% and 0.61% to 2.39%,respectively.85 clinical samples were tested by the established TaqMan-based Real-time PCR method and the conventional PCR method,the positive rate were 7.06%(6/85)and 5.88%(5/85),respectively.All PCR positive samples were also tested positive by TaqMan-based Real-time PCR method,the coincidence rate was 100%.The TaqMan-based Real-time PCR method provided a useful method for molecular epidemiological survey of DAdV-A.