非洲猪瘟病毒荧光PCR检测方法的建立及评价  

Development and evaluation of the fluorescence quantitative PCR assay for detection of African swine fever virus

作　　者：陈小金 朱向东 李文钢[2] 赵丹 张俊哲 李秀梅[2] 董志珍 张霞[3] CHEN Xiao-jin;ZHU Xiang-dong;LI Wen-gang;ZHAO Dan;ZHANG Jun-zhe;LI Xiu-mei;DONG Zhi-zhen;ZHANG Xia(Animal and Plant and Foodstuffs Inspection Center,Tianjin Customs,Tianjin 300457,China;Tianjin Animal Disease Prevention and Control Center,Tianjin 300402,China;Henry Fok College of Food Science,Shaoguan University,Shaoguan 512005,China)

机构地区：[1]中华人民共和国天津海关动植物与食品检测中心,天津300457 [2]天津市动物疫病预防控制中心,天津300402 [3]韶关学院英东食品学院,广东韶关512005

出　　处：《中国兽医科学》2022年第7期815-823,共9页Chinese Veterinary Science

基　　金：天津市科技计划项目(19YFZCSN00370);海关总署科研项目(2019HK017);广东省普通高校重点领域专项(2020-ZDZX1042)。

摘　　要：为建立非洲猪瘟病毒(ASFV)荧光PCR方法并研制配套检测试剂,本研究以非洲猪瘟病毒E183L基因为靶基因,运用TaqMan探针法建立检测方法,并对其进行验证评价。根据E183L基因序列设计特异性引物和探针,将目的片段克隆到pBackZero-T载体,并转化至大肠杆菌JM109中制备标准品,并绘制标准曲线。同时对本方法的灵敏度、特异性、重复性和实际样品检测效果进行评价,并由WOAH非洲猪瘟参考实验室对该方法和试剂进行验证。结果显示,本研究以3.3×10^(7)~3.3×10^(1) copies/μL浓度范围内的质粒为标准品建立标准曲线;所建立的方法与其他几种常见猪病病毒无交叉反应;最低检测下限为3.3 copies/μL;批内和批间变异系数均小于2%;对WOAH参考实验室阳性样品的评估试验表明,本研究建立的方法与参考实验室推荐方法的符合率为93.8%。运用该方法检测国内猪场、无害化处理厂、屠宰厂和市售样品34350份,检出环境阳性81份,检出率略高于WOAH推荐方法。上述结果表明,本试验所建立的方法适用于血液、拭子、猪肉制品以及环境样品中ASFV检测和猪场监控检测,可作为ASFV的诊断及分子流行病学调查的技术手段。Based on the E183L gene of ASFV reference strain,the fluorescence PCR method was established and the detection reagent was developed,then the verification and evaluation for the method was performed.A pair of specific primers and probe were designed according to the sequence of E183L gene.The plasmid was constructed based on the target fragment and pBackZero-T vector,which aims to preparae the standard plasmid and standard curve.The sensitivity,specificity and repeatability of this study were evaluated,meanwhile,the method and reagent were verified by WOAH African swine fever reference laboratory.Within the plasmid concentration range of 3.3×10^(7)—3.3×10^(1 )copies/μL,the standard curve was drawn.The established method has no cross reaction with other porcine disease viruses.The detection limit was 3.3 copies/μL,while the intra-and inter-assay coefficients of variation were less than 2%.The pool of positive samples was detected with the method recommended by the WOAH reference laboratory and the method established in this study,the coincidence rate was 93.8%.34350 samples that from pig farms,rendering house,slaughterhouses and pork products were detected by this method,and 81 environmental samples were positive,which suggests the positive detection rate is slightly higher than that recommended by WOAH.The result shows that the method established in this paper is suitable for the detection of ASFV in blood,swabs,pork products and environmental samples,also suitable for epidemic situation monitoring in pig farms.It can be used as a technical means for the diagnosis and molecular epidemiological investigation of ASFV.

关 键 词：非洲猪瘟病毒 荧光PCR 方法评价 

分 类 号：S852.659.1[农业科学—基础兽医学]