对虾IHHNV荧光定量PCR检测方法的建立  

Development of a Real-time TaqMan-quantitative PCR Assay for IHHNV Detection

作　　者：谢丽基[1] 谢芝勋[1] 庞耀珊[1] 卢兆发 谢志勤[1] 刘加波[1] 邓显文[1] 唐小飞[1] 

机构地区：[1]广西兽医研究所,广西南宁530001 [2]广西水产畜牧局,广西南宁530022

出　　处：《水生态学杂志》2008年第6期132-135,共4页Journal of Hydroecology

基　　金：广西科技攻关项目(桂科攻0322006-3A)

摘　　要：根据GenBank中对虾传染性皮下和造血器官坏死病毒(IHHNV)保守基因序列(AF218226),设计合成了1对引物和1条TaqMan探针,建立了检测IHHNV的荧光定量PCR技术。将建立的荧光定量PCR检测方法与常规PCR对比。结果显示,所建立的荧光定量PCR方法灵敏度可达2个拷贝,比常规PCR灵敏度高1000倍。对保存的15份经常规PCR检测为IHHNV阳性的DNA样品进行荧光定量PCR检测,结果都为阳性,检测的病毒含量为2.15×107～4.21×104拷贝/μL。用该方法对不同浓度的样品进行了重复检测,表明该方法具有良好的重复性,可满足IHHNV的临床诊断需要。A pair of primers and a TaqMan probe were designed and synthesized according to the conserved gene sequences of Infectious Hypodermal and Haematopoietic Necrosis （IHHNV） in GenBank （AF218226）, and then reaction parameters were optimized to develop a real -time TaqMan -quantitative PCR assay. The developed quantitative PCR assay was compared with that of routine PCR. This quantitative PCR assay could detect 2 template copies of plasmid DNA, and its sensitivity was 1 000 times higher than that of the routine PCR. The real - time Taq- Man - quantitative PCR results of 15 routine PCR positive clinical samples showed that concentration of the clinical samples were 2.15 ×10^7 -4.21 × 10^4 copies/μL. The samples were examined using the quantitative PCR repeatedly and the results indicated that the quantitative PCR was reproducible and could be used successfully for the diagnosis of IHHNV infection.

关 键 词：对虾 IHHNV病毒 PCR 检测 

分 类 号：S945.19[农业科学—水产养殖]