H3N2亚型猪流感病毒实时荧光定量PCR快速检测方法的建立  

Development of a real-time fluorescent quantitative PCR assay for rapid detection of swine influenza virus H3N2 subtype

作　　者：陈艳[1] 张春明[1] 乔传玲[1] 杨焕良[1] 张飞[1] 吴运谱[1] 辛晓光[1] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室农业部动物流感重点开放实验室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2009年第10期894-899,共6页Chinese Veterinary Science

基　　金：国家科技攻关计划项目(2004BA519A55)

摘　　要：根据H3N2亚型猪流感病毒(SIV)血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)基因保守序列,分别设计并合成了特异性引物和TaqMan MGB探针,利用实时荧光定量PCR技术检测SIV。使用含有选定检测序列的重组质粒pMD18-H3HA、pMD18-N2NA标准品绘制标准曲线,其相关系数分别为0.99015(H3HA)和0.99947(N2NA)。检测结果显示,该方法的敏感性可达100 copies/μL,除H3N2亚型SIV外,对H1亚型、H9亚型SIV以及猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪瘟病毒、猪生殖与呼吸综合征病毒和猪2型圆环病毒的检测均为阴性,应用该方法对实验室感染样品进行检测,其结果与病毒分离结果的符合率为95.83%。表明,该方法特异性强、重复性好,有望成为H3N2亚型SIV的一种特异、敏感、快速的定量检测方法。The specific primers and TaqMan MGB probes were designed according to the conserved region of H3HA and N2NA genes of swine influenza virus（SIV） H3N2 subtype. A real-time fluorescent quantitative PCR（RF-PCR） assay was developed for detection of SIV H3N2 subtype. A series of dilutions of recombinant plasmids including pMD18 H3HA and pMD18-N2NA were prepared and used to generate standard curves. The results showed that the RF-PCR was capable of detecting 100 copies of HA or NA genes per microliter,with a correlation coefficient of 0. 990 15（H3HA） and 0. 999 47（N2NA） ,respectively. The results were negative for the detection of transmissible gastroenteritis virus,porcine epidemic diarrhea virus,classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and SIV H1 and H9 subtypes. The coincidence rates between RF-PCR and virus isolation were 95.83%. The method was highly specific and sensitive,and could be used for rapid quantitative detection of SIV H3N2 subtype.

关 键 词：猪流感病毒 H3N2亚型 TAQ MAN MGB探针 实时荧光定量PCR 

分 类 号：S852.659.5[农业科学—基础兽医学]