分子信标荧光定量PCR检测H3N2亚型猪流感病毒方法的建立  

Establishment of a Molecular Beacon Real Time Fluorescent Quantitative PCR Assay for Detection of Swine Influenza Virus H3N2 Subtype

作　　者：禹思宇[1] 周宇[2] 唐连飞[1] 孟芳[1] 袁晓芬[1] 梁斌[1] 

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

出　　处：《中国动物检疫》2015年第7期67-71,76,共6页China Animal Health Inspection

基　　金：国家质检总局科技资助项目(2014IK243)

摘　　要：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(SIV)的HA和NA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,利用实时荧光定量PCR技术检测H3N2亚型SIV。构建重组质粒p SK-H3和p SK-N2并绘制标准曲线。结果显示,该检测方法的敏感性达到102拷贝/μL;与其它主要相关病毒均不发生交叉反应,批内和批间试验的重复性变异系数(CV)均小于3%,表明该方法灵敏度强、特异性好。此方法的建立将为流感病毒H3N2亚型定型检测提供一种快速有效的方法。A new assay for the detection of swine influenza virus（SIV）was developed with a novel nucleic acid probe—molecular beacon. The specific primers and molecular beacon probes were designed according to the conserved region of H3 and N2 genes of swine influenza virus（SIV）H3N2 subtype. A real-time fluorescent quantitative PCR as-say was developed for detection of SIV H3N2 subtype. A series of dilutions of recombinant plasmids including pSK-H3 and pSK-N2 were prepared and used to generate standard curves. Under the optimized reaction conditions,the results showed that the developed assay was specific for detecting SIV with a detection limit of 102 copies without cross-reac-tions to other swine viruses.The repeatability tests indicated that the inter-and intra-variation were less than 3%.The method was highly specific and sensitive,providing an effective method for detection of swine influenza virus H3N2 subtype.

关 键 词：猪流感病毒 H3N2亚型 分子信标探针 实时荧光定量PCR 

分 类 号：S852.651[农业科学—基础兽医学]