H1、H3亚型猪流感病毒双重实时荧光定量RT-PCR检测方法的建立及应用  

Development and Application of Duplex Real-time RT-PCR Assay for Detection of H1 and H3 Subtype Swine Influenza Viruses

作　　者：王博[1,2,3] 王慧煜[1] 赵宝华[3] 罗静[2] 王承民[2] 何宏轩[2] 韩雪清 

机构地区：[1]中国检验检疫科学研究院,北京100029 [2]中国科学院动物研究所,北京100101 [3]河北师范大学,石家庄050024

出　　处：《中国畜牧兽医》2017年第10期3035-3041,共7页China Animal Husbandry & Veterinary Medicine

基　　金：十二五国家科技支撑计划课题(2013BAD12B01);国家林业局野生动物疫源疫病监测项目;中国科学院战略生物资源科技支撑体系运行专项野生动物疫源疫病样品组织库(CZBZX-1)

摘　　要：为建立一种快速、准确地检测H1和H3亚型猪流感病毒(swine influence virus,SIV)的方法,根据H1和H3亚型SIV血凝素(hemagglutinin,HA)基因保守序列,分别设计2对特异性引物和2条Taq Man探针,建立双重实时荧光定量RT-PCR方法。结果显示,该方法敏感性高,可检测到最低拷贝数为102拷贝/μL;重复性良好,重复孔Ct值的变异系数均在5%以下;特异性好,除H1和H3亚型SIV外,H4、H5、H7、H9亚型SIV、猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒检测均为阴性;在田间样品中成功检出1株H1和1株H3亚型SIV,检出率为1.16%。该方法准确、快速、灵敏、特异,可为H1、H3亚型SIV的快速鉴别检测及流行病学调查提供有效的技术手段。To establish a rapid,accurate method to diagnose and detect H1 and H3 subtype swine influenza viruses（ SIV） at the same time,the specific primers and Taq Man probes were designed according to the conserved region of the HA gene of H1 and H3 subtype SIV. A duplex Real-time RT-PCR assay was developed for detection of H1 and H3 subtype SIV. The results showed that the Real-time RT-PCR could detect 102copies/μL of H1 and H3 subtype SIV,the sensibility was well. Coefficient of variation of Ct value between repeating groups were all below 5%,the repeatability was favorable. The results were negative for the detection of H4,H5,H7,H9 subtypes SIV,classical swine fever virus,porcine reproductive and respiratory syndrome virus,foot and disease virus and pseudorabies virus,the specificity was fine.One sample was H1 subtype SIV,and one sample was H3 subtype SIV,by the established assay,the positive rate was 1.16%. The method was highly accurate,rapid,sensitive and specific,and could provide a method for rapid detection and epidemiological investigation of H1 and H3 subtype SIV.

关 键 词：H1和H3亚型猪流感病毒(SIV) 双重实时荧光定量RT-PCR TAQMAN探针 

分 类 号：S852.65[农业科学—基础兽医学]