微滴式数字PCR定量检测禽脑脊髓炎病毒方法的建立  

Establishment and application of droplet digital PCR for quantitative detection of avian encephalomyelitis virus

作　　者：谢志勤 谢芝勋 张艳芳 范晴 谢丽基 万丽军 罗思思 李孟 张民秀 曾婷婷 黄娇玲 王盛 李丹 韦悠 李小凤 任红玉 阮志华 XIE Zhiqin;XIE Zhixun;ZHANG Yanfang;FAN Qing;XIE Liji;WAN Lijun;LUO Sisi;LI Meng;ZHANG Minxiu;ZENG Tingting;HUANG Jiaoling;WANG Sheng;LI Dan;WEI You;LI Xiaofeng;REN Hongyu;RUAN Zhihua(Key Laboratory of China(Guangxi)-ASEAN Cross-Border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of China,Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning 530001,China)

机构地区：[1]广西壮族自治区兽医研究所广西兽医生物技术重点实验室农业农村部中国-东盟(广西)跨境动物疫病防控重点实验室,广西南宁530001

出　　处：《中国兽医学报》2023年第4期668-673,719,共7页Chinese Journal of Veterinary Science

基　　金：广西重点研发计划资助项目(桂科AB16380003);广西基地与人才专项资助项目(AD17195083);广西创新驱动专项资助项目(AA17204057);国家“万人计划”领军人才专项资助项目(W02060083);“广西八桂学者”专项资助项目(2019A50)。

摘　　要：旨在建立微滴式数字PCR(ddPCR)检测禽脑脊髓炎病毒(avian encephalomyelitis virus,AEV)的方法,并实现对AEV绝对定量检测。根据已发表的AEV的VP1基因为靶序列,在其保守区域内设计了特异性扩增的引物和探针序列,应用合成的引物和探针建立ddPCR检测AEV方法,并测定其特异性、灵敏性和重复性。结果显示,建立方法的最佳引物终浓度为1.0μmol/L,探针终浓度为0.5μmol/L,退火温度为58℃。特异性测试结果,本方法只检出AEV,没有检出禽流感病毒、鸡新城疫病毒、鸡传染性支气管炎病毒、禽呼肠孤病毒、禽偏肺炎病毒和鸡传染性喉气管炎病毒,且无交叉反应。灵敏性结果显示,本方法对pMD18-AEV重组质粒DNA最低检测限为5.9拷贝/μL,敏感性高。用4个连续稀释的pMD18-AEV重组质粒DNA的拷贝数的对数值与ddPCR检测的拷贝数的对数值绘制绝对定量曲线,其曲线呈良好的线性关系(R2=0.9994),结果稳定。3次重复检测结果的变异系数均小于5%,重复性好。对采集的病鸡临床样品65份进行ddPCR和荧光定量PCR检测,结果显示2种方法AEV阳性检出率均为4.62%(3/65)。结果表明,本研究建立的ddPCR方法检测AEV特异性高、灵敏好和稳定可靠,为绝对定量检测AEV提供了技术手段,对有效防控AEV有重要意义。The objective of this study was to develop and evaluate a droplet digital PCR(ddPCR)method for absolute quantitative detection avian encephalomyelitis virus(AEV).According to the published VP1gene sequence of AEV,a set of specific oligonucleotide primers and probes were designed in its conservative region,and the ddPCR method for detecting AEV was established using the synthesized primers and probes,and its specificity,sensitivity and repeatability were determined.The results showed that the optimal final concentration of primer was 1.0μmol/L and final concentration of probe was 0.5μmol/L.The annealing temperature was 58℃.The specificity test results showed that this method only detected AEV,but did not detect avian influenza virus,Newcastle disease virus,avian infectious bronchitis virus,avian reovirus,avian metapneumoniae virus and avian infectious laryngotracheitis virus.There was no cross reaction.The limit of detection was 5.9copies/μL of pMD18-AEV,indicating that it had good sensitivity.The absolute quantitative curve was drawn with the logarithm of the copy number of four continuously diluted pMD18-AEV and the copy number detected by ddPCR.The curve showed a good linear relationship(R2=0.9994).The results indicated that it was stable.The coefficients of variation of three repeated tests were all less than 5%.It showed that the repeatability was good.Sixty five clinical samples collected from sick chickens were tested by ddPCR and fluorescence quantitative PCR test.The results showed that the positive detection rate of AEV by both methods were 4.61%(3/65).In conclusion,this ddPCR method had high specificity,good sensitivity,stability and reliability for the detection of AEV,which provided technical means for the absolute quantitative detection of AEV and had important significance for prevention and control of AEV.

关 键 词：微滴式数字PCR 禽脑脊髓炎病毒 VP1基因 

分 类 号：S852.65[农业科学—基础兽医学]