美国白蛾核型多角体病毒实时荧光定量PCR检测方法的建立及应用  

Establishment and application of real-time fluorescence quantitative PCR for detection of Hyphantria cunea nucleopolyhedrovirus

作　　者：乔鲁芹[1] 曲良建[2] 王玉珠[2] 张永安[2] 杨忠岐[2] 陶万强 关玲 

机构地区：[1]山东农业大学植物保护学院,山东泰安271018 [2]中国林业科学研究院森林生态环境与保护研究所,北京100091 [3]北京市林业保护站,北京100029

出　　处：《昆虫学报》2010年第7期824-830,共7页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(30671688);国家“十一五”科技支撑计划课题(2006BAD08A1202);北京市科委项目(Z0006344040191)

摘　　要：为了建立对昆虫核型多角体病毒(nucleopolyhedrovirus)进行定量检测方法,本研究选用美国白蛾Hyphantria cunea核型多角体病毒的polyhedrin基因为目的基因,经引物设计、PCR扩增、目的片段与载体连接转化以及重组质粒的鉴定,并对重组质粒标准品和样品DNA进行检测,构建出美国白蛾核型多角体病毒SYBR GreenⅠ荧光定量标准曲线。统计学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(R=0.998)。该方法的检测灵敏度为101～102拷贝/μL,线性范围达5个数量级,扩增产物形成单一的特异性熔解峰,组内组间的变异系数均小于3%。根据已建立的方法对不同感染时间段的感病幼虫进行检测,每毫克幼虫样本内病毒polyhedrin基因拷贝数增殖倍数对数值与感染时间(d)呈正相关(R=0.987)。这些结果表明,建立的美国白蛾核型多角体病毒荧光定量PCR检测方法是可靠的,具有灵敏度高、重复性好等特点,可为昆虫核型多角体病毒体内增殖动态研究及昆虫病毒的标准化生产提供参考。Primers were designed based on polyhedrin from Hyphantria cunea nucleopolyhedrovirus in order to establish the detection method of SYBR GreenⅠ real-time fluorescence quantitative PCR for HcNPV, and the standard curve of SYBR GreenⅠreal-time fluorescence quantitative PCR for polyhedrin was established through PCR amplification, ligation of target genes and vector, transformation, identification of recombinant plasmid and DNA sample detection. Statistic analysis showed that there was a good linear relationship between Ct value and the logarithmic value of plasmid concentrations （R=0.998）. The sensitivity of the method was 101-1×102 copies/μL, and wide detection range of 5 orders of magnitude was obtained. Larvae 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 （R= 0.987）. The results show that the detection method for HcNPV is of high sensitivity, reproducibility and credibility, which will facilitate the epizootiology and the standardized production for insect virus.

关 键 词：美国白蛾核型多角体病毒 SYBR GreenⅠ 荧光定量PCR 感病幼虫 检测 

分 类 号：Q966[生物学—昆虫学]