猪流行性腹泻病毒锁核酸探针荧光定量PCR检测方法的建立  

Methodology Development of a Locked Nucleic Acidbased Fluorescent Quantitation RTPCR for Detecting Porcine Epidemic Diarrhea Virus

作　　者：李艳 卞志标 翟少伦 李春玲 LI Yan;BIAN Zhibiao;ZHAI Shaolun;LI Chunling(Institute of Animal Health,Guangdong Academy of Agricultural Sciences/Key Laboratory of Livestock Disease Prevention of Guangdong Provincel/Scientific Observation and Experiment Station of Veterinary Drugs and DiagnosticTechniques of Guangdong Province,Ministry of Agriculture,Guangzhou Guangdong 510640;Maoming Branch center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology,Maoming Guangdong 525000)

机构地区：[1]广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站,广东广州510640 [2]岭南现代农业科学与技术广东省实验室茂名分中心,广东茂名525000

出　　处：《广东畜牧兽医科技》2024年第2期30-36,共7页Guangdong Journal of Animal and Veterinary Science

基　　金：广东省重点领域研发计划项目(2020B0202080004);广州市重点研发项目(202206010192);广东省科技计划项目(2023B0202010009);广东省农业科学院大广食品专家工作站建设项目(2023工作站16);江门市科技计划项目(江科[2021]183号);广东省农业农村厅科技计划项目(2023KJ119)。

摘　　要：为建立一种快速、灵敏的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)早期痕量检测方法,该研究以PEDV的M基因为靶基因,设计了特异性引物和锁核酸(Locked Nucleic Acid,LNA)-TaqMan探针,经过条件优化,建立了基于LNA-TaqMan探针的PEDV荧光定量PCR方法,并与常规TaqMan探针法进行了比对。结果显示,所建立的PEDV的LNA-Taq-Man探针荧光定量PCR方法最低检测限为3.2拷贝/微升,较常规TaqMan探针法提高了10倍;与猪传染性胃肠炎病毒、猪轮状病毒和猪德尔塔冠状病毒等多种病原不存在交叉反应,特异性良好;批内和批间重复性结果的变异系数均小于1%,重复性较好。应用该方法对103份临床样品进行检测,结果显示,共检出PEDV阳性样品47份,阳性率45.63%,与常规TaqMan探针法检测结果的阳性符合率为90.38%。本研究建立的基于LNA-TaqMan探针的荧光定量PCR方法为PEDV的精准检测和流行病学调查提供了一种新的技术选择。In this study,a LNA-based fluorescent quantitation PCR method was developed for the rapid,accurate and trace detection of porcine epidemic diarrhea virus(PEDV)by targeting the M gene.Special primers and LNA-TaqMan probe were designed according to the M gene,and the reaction conditions have been optimized.The LNA-based fluorescent quantitation PCR method was compared with the conventional TaqMan probe method.Results showed that the detection limit of the method was 3.2 copies/μL,improved by 10 times compared to conventional TaqMan probe method.The method was specific of PEDV and no cross reaction to TGEV,PoRV,and PDCoV.The method had a good repeatability with variation coefficient of intra-and inter-group less than 1%.The method was used to detect 103 clinical samples,the results showed that 47 samples were positive for PEDV,with a positive rate of 45.63%,and the positive coincidence rate with the conventional TaqMan probe PCR assay was 90.38%.This LNA-based fluorescent quantitation PCR method provides a new technical for accurate detection and epidemiological investigation of PEDV.

关 键 词：猪流行性腹泻病毒 M基因 LNA-TaqMan探针荧光定量PCR 

分 类 号：S852.651[农业科学—基础兽医学]