摘要
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。
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 Realtime 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 par-vovirus,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 interassay 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.
作者
万春和
刘荣昌
程龙飞
施少华
傅光华
陈红梅
傅秋玲
黄瑜
WAN Chunhe;LIU Rongchang;CHENG Longfei;SHI Shaohua;FU Guanghua;CHEN Hongmei;FU Qiuling;HUANG Yu(Fujian Animal Disease Control Technology Development Center,Institute of Animal Husbandryand Veterinary Medicine,Fujian Academy of Agricultural Science,Fuzhou 350013,China)
出处
《中国畜牧兽医》
CAS
北大核心
2018年第5期1341-1348,共8页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31602068)
国家水禽产业技术体系(CARS-42)
福建省农业科学院兽医青年科技创新团队(STIT2017-3-10)
福建省农业科学院畜牧兽医研究所基金(MYQJ2015(S)-3、MYQJ2016(G)-1)
福建省农业科学院青年科技英才项目(YC2015-12)
福建省属公益类项目(2018R1023-5)