数字RT-PCR技术检测H3N2亚型猪流感病毒方法的建立  

Establishment of Digital RT-PCR Assay for Detection of Swine Influenza Virus H3N2 Subtype

作　　者：唐连飞[1] 禹思宇[1] 周宇[2] 

机构地区：[1]湖南出入境检验检疫局检验检疫技术中心,长沙410004 [2]惠州出入境检验检疫局,惠州516006

出　　处：《中国畜牧兽医》2017年第6期1847-1853,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家质检总局科技资助项目(2014IK243)

摘　　要：试验旨在利用新型荧光探针——分子信标,建立一种检测猪流感病毒的新方法。根据H3N2亚型猪流感病毒(swine influenza virus,SIV)的H3和N2基因的保守基因序列,分别设计并合成了特异性引物和分子信标探针,利用数字RT-PCR技术检测H3N2亚型SIV。结果显示,该数字RT-PCR检测方法与其他主要相关病毒均不发生交叉反应,重复性良好;对猪H3N2流感病毒而言,最低可检测到106倍稀释的病毒株。数字RT-PCR方法能够对RNA模板定量分析。在H3N2SIV的鉴定上,数字RT-PCR方法较实时荧光定量PCR方法更灵敏、准确。A new assay for the detection of swine influenza virus （SIV） was developed with a novel nucleic acid probe——Molecular beacon in this study. The specific primers and molecular beacon probes were designed according to the conserved region of H3 and N2 genes of SIV H3N2 subtype. A digital RT-PCR assay was developed for detection of SIV H3N2 subtype. The results showed that SIV H3N2 subtype could be identified simultaneously on this microarry with high sensitivity and reproducibility,which could reach to 106 dilute viruse. The conclusion was that the digital RT-PCR method could analyze quantitatively the RNA templates.On the identification of H3N2 SIV,the digital RT-PCR method was much more scientific than Real-time quantitative PCR method.

关 键 词：猪流感病毒 H3N2亚型 分子信标探针 数字RT-PCR 实时荧光定量PCR 灵敏性 特异性 重复性 

分 类 号：S852.659.5[农业科学—基础兽医学]