茶尺蠖核型多角体病毒荧光定量PCR检测方法的建立  

Development of the Fluorescent Quantitative PCR for Detection of EoNPV in Ectropis Oblique

作　　者：杜军利[1,2] 张传溪[2] 肖强[1] 付建玉[1] 殷坤山[1] 

机构地区：[1]中国农业科学院茶叶研究所,浙江杭州310008 [2]浙江大学昆虫科学研究所,浙江杭州310029

出　　处：《茶叶科学》2010年第3期203-207,共5页Journal of Tea Science

基　　金：国家科技支撑计划子课题(2008BADA5B06-3);科技基础性工作专项(2008FY210500)

摘　　要：根据茶尺蠖核型多角体病毒(Ectropis oblique nucleopoly hedrovirus,EoNPV)基因序列设计一对引物,并从感染该病毒的虫体中提取EoNPV基因组DNA,进行PCR扩增,将该基因片段与载体pMD18-T连接,转化入大肠杆菌TG1中,经单克隆菌落筛选及测序鉴定后得到重组质粒,以该质粒为荧光定量PCR标准品模板稀释后建立标准曲线。构建的标准曲线线性关系良好,相关系数为0.989,检测范围为103～108拷贝/μL,特异性和重复性较好。该方法可以准确地判断出病毒拷贝数,为茶尺蠖核型多角体病毒在虫体内增殖动态的研究和病毒制剂的质量检测提供了方法依据。A pair of primer was designed according to the Ectropis oblique nucleopolyhedrovirus （EoNPV） genome DNA sequence. Virus DNA was extracted from the tea looper larvae, Ectropis oblique which were infected with the EoNPV. The DNA fragment was amplified, then cloned into pMD18-T vector and transferred into E. coli TG1. A single clone was selected and sequenced, and the extracted recombinant plasmid DNA was used as a positive quantitative template to establish a standard curve. The standard curve showed a linear relationship between cycle threshold （CT） and template concentration ranging from 10^3～10^8 copies/μL with a correlation coefficient of 0.989, and the quantitative PCR was more repeatable and specific than traditional PCR. The fluorescent quantitative PCR method for detecting EoNPV was developed, providing a basis for the research of EoNPV replicating in the host body and quantitative detection of the EoNPV in biopesticide products.

关 键 词：茶尺蠖 核型多角体病毒 荧光定量PCR 检测 

分 类 号：S435.711[农业科学—农业昆虫与害虫防治]