猪流感病毒TaqMan荧光定量RT-PCR检测方法的建立  

Establishment of real-time fluorescence quantitative RT-PCR detection method for swine influenza virus

作　　者：黄书 梁海英[1] 曾智勇[1] 汤德元[1] 王彬[1] 叶泥 边孟婷 柳佳佳 潘向英 田红利 HUANG Shu;LIANG Haiying;ZENG Zhiyong;TANG Deyuan;WANG Bin;YE Ni;BIAN Mengting;LIU Jiajia;PAN Xiangying;TIAN Hongli(College of Animal Science,Guizhou University,Guiyang550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025

出　　处：《中国兽医学报》2024年第4期651-656,共6页Chinese Journal of Veterinary Science

基　　金：贵州省科技支撑计划资助项目(黔科合支撑[2021]一般162项目)。

摘　　要：参照GenBank登录的猪流感病毒(swine influenza virus,SIV)基质蛋白(matrix protein,M)基因保守序列设计1对特异性引物和1个TaqMan探针,构建重组质粒作为绝对定量模板,通过优化反应体系及条件建立了检测SIV的荧光定量RT-PCR方法,测试了该方法的特异性、灵敏度及重复性。结果显示,建立的方法与猪瘟病毒、猪蓝耳病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪圆环病毒2型、猪伪狂犬病病毒、猪细小病毒和猪乙型脑炎病毒等不存在交叉反应,特异性强;最低检测限度为1.0拷贝/μL,比普通RT-PCR方法灵敏10倍;组内和组间重复性试验的变异系数分别在2.2%和1.9%以下,重复性较好;应用该方法对220份临床疑似猪流感样品进行检测,荧光定量RTPCR方法和普通RT-PCR检测率分别为2.7%和1.8%,阳性符合率为100%。结果表明,建立的TaqMan荧光定量RT-PCR方法特异性强、灵敏度高、重复性好,可快速检测样品中的各种SIV,从而更好地诊断和监测猪流感。In order to establish an efficient,sensitive and specific method to accurately detect swine influenza virus(SIV),specific primers and a TaqMan probe were designed according to the conserved sequence of SIV viral matrix protein(M)gene registered in GenBank,and the recombinant plasmid was constructed as an absolute quantitative template sensitivity and repeatability.The results showed that the established method had no cross reaction with classical swine fever virus,porcine reproductive and respiratory syndrome virus,porcine epidemic diarrhea virus,transmissible gastroenteritis virus,porcine circovirus type 2,porcine pseudorabies virus,porcine parvovirus and porcine Japanese encephalitis virus,and had strong specificity.The minimum detection limit was 1.0copies/μL,10times more sensitive than ordinary RT-PCR methods.The coefficient of variation of intra group and inter group repeatability tests were below 2.2%and 1.9%,respectively,indicating good repeatability.The method was applied to detect 220clinical suspected swine flu samples,and the detection rates of fluorescence quantitative RT-PCR and conventional RT-PCR were 2.7%and 1.8%,respectively,with a positive coincidence rate of 100%.The results indicate that the established TaqMan fluorescence quantitative RT-PCR method has strong specificity,high sensitivity,and good repeatability,and can quickly detect various SIVs in samples,thereby better diagnosing and monitoring swine influenza.

关 键 词：猪流感病毒 TAQMAN探针 荧光定量RT-PCR方法 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]