茶尺蠖核型多角体病毒(EoNPV)实时荧光定量PCR检测方法的优化及应用  

Optimization and Application of Real-time Fluorescence Quantitative PCR for Detection of Ectropis obliqua Nucleopolyhedrovirus

作　　者：袁志军[1] 张传溪[2] 肖强[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]浙江大学应用昆虫学研究所,浙江杭州310058

出　　处：《茶叶科学》2013年第3期229-236,共8页Journal of Tea Science

基　　金：国家"十二五"科技支撑计划课题(2011BAD01B02)

摘　　要：选用茶尺蠖核型多角体病毒(EoNPV)基因组中序列特异性很高的p16基因为目标基因,通过设计引物、PCR扩增、目的片段与载体连接转化获得含p16基因的重组质粒进行测序,进而以经测序鉴定的p16基因的PCR纯化产物作为实时荧光定量PCR标准品模板,稀释后建立SYBR Green I荧光定量标准曲线,统计分析显示标准品浓度的对数值与Ct值之间存在良好的线性关系(R2=0.9981)。该方法的检测灵敏度为102,扩增产物形成单一的特异性熔解峰,组内组间变异系数均小于3%。同时分别对含有其他茶树害虫病毒和不同浓度的茶尺蠖病毒产品的样品进行检测,能明确样品中是否含有EoNPV及含量。根据已建立的方法对感染病毒后不同时间段的茶尺蠖幼虫进行检测,每克幼虫样本内p16基因拷贝数的增殖倍数对数值与感染时间呈正相关(R2=0.9935),可以获得茶尺蠖幼虫感染病毒后不同时间的增殖动态。上述结果表明,该方法能定性定量鉴定茶尺蠖核型多角体病毒,可用于茶尺蠖核型多角体病毒类生物农药的检测和感染过程检测。Primers were designed based on p16 gene from optimize the detection method of SYBR Green I real-time Ectropis obliqua nucleopolyhedrovirus in order to fluorescence quantitation PCR for EoNPV, and the standard curve of SYBR Green I real-time fluorescence quantitative PCR for p16 gene was established using the recombinant plasmid DNA as template. Statistic analysis showed that there was a good linear relationship between Ct value and the logarithmic value of plasmid concentrations （R2=0.9981）. The sensitivity of the method was 102 copies/ktl, and wide detection range of 6 orders of magnitude was obtained. Larave infected at different time were sampled and detected by the method, and the results showed that there was a good linear relation between the logarithmic value of the multiples of gene copies and the infection time （R2=0.9935）. Experimental results indicated that the method can identify biological pesticides from qualitative and quantitative aspects, and distinguish the highhomology nuclearpolyhedrosisvirus （Euproctis pseudoconspersa nuclearpolyhedrosisvirus） from EoNPV accurately.

关 键 词：EoNPV SYBR GreenⅠ 定性定量 检测 

分 类 号：S435.711[农业科学—农业昆虫与害虫防治]