猪伪狂犬病毒野毒株SYBR GreenⅠ实时荧光定量PCR诊断试剂盒的研制  

Development of SYBR GreenⅠ Real-time Quantitative PCR Kit for Detection of PRV Wild-type Strains

作　　者：余波[1] 周思旋[1] 谭诗文[1] 徐景峨[1] 史开志[1] 杨莉[1] 

机构地区：[1]贵州省畜牧兽医研究所,贵州贵阳550005

出　　处：《河南农业科学》2014年第6期128-131,144,共5页Journal of Henan Agricultural Sciences

基　　金：贵州省科技厅农业攻关项目([2010]3085);贵州省畜禽健康养殖技术创新能力建设项目([2010]4004)

摘　　要：根据GenBank中猪伪狂犬病毒(PRV)gE基因序列设计特异性的引物,PCR扩增获得PRV gE基因片段,构建重组质粒pMD18-T-gE,作为阳性标准品。对SYBR GreenⅠ实时荧光定量PCR反应条件进行优化,建立猪PRV SYBR GreenⅠ实时荧光定量PCR诊断方法,研制诊断试剂盒。扩增产物的熔解曲线分析结果显示只出现单特异峰,无引物二聚体,对猪圆环病毒(PCV-2)、猪细小病毒(PPV)、PRV(gE基因缺失株)、猪源E.coli、猪瘟病毒(CSFV)、猪繁殖与呼吸障碍综合征病毒(PRRSV)均无阳性信号扩增,且重复性好,灵敏度可达2.3×101拷贝/μL。结果表明,研制的PRV野毒株SYBR GreenⅠ实时荧光定量PCR试剂盒特异、灵敏、快速、重复性好,适于伪狂犬病毒临床样品的检测。According to the gene sequences of gE gene of PRV in GenBank,one pairs of specific primer was designed for amplifying the specific fragments of gE gene. Then gE gene of PRV amplified by PCR was cloned into pMD18-T vector and it was used as positive standard. After optimization of annealing temperature and primers concentrations, a SYBR Green Ⅰ real-time quantitative PCR kit was developed for detection of PRV wild-type strains. The melting curve analysis using SYBR Green Ⅰ dye showed one specific peak,and primer-dimers peak was not observed. No fragment was amplified from PRV strains depleted of gE gene, PPV, PCV-2, Escherichia coli, CSFV, PRRSV by the SYBR Green Ⅰ real-time quantitative PCR. The PCR kit was highly sensitive in 2.3 × 10^1 copies/μL DNA. The results revealed that the PCR kit was sensitive, specific and it could be used to detect PRV wild-type strains in clinical samples.

关 键 词：猪伪狂犬病毒 野毒株 GE基因 荧光定量PCR 诊断试剂盒 

分 类 号：S854.43[农业科学—临床兽医学]