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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。
In order to establish a method for the quantitative detection of the nucleopolyhedrovirus, we selected the polyhedrin gene of the Hyphantria cunea nuclear polyhedrosis virus as the target gene. After the primers were designed and amplified by PCR, the target fragment was ligated with the vector Transformation and identification of the recombinant plasmids. The recombinant plasmid standard and sample DNA were detected, and the SYBR Green Ⅰ fluorescence quantitative standard curve of the American white moth nuclear polyhedrosis virus was constructed. Statistical analysis showed a good linear relationship between the logarithmic value of the standard concentration and the Ct value (R = 0.998). The detection sensitivity of this method was 101-102 copies / μL with a linear range of 5 orders of magnitude. The amplification products formed a single specific melting peak, and the coefficient of variation (CV) between the two groups was less than 3%. According to the established method, the larvae of susceptible larvae at different infection stages were tested. The logarithm of multiplication multiples of virus polyhedrin gene per milligram larva was positively correlated with the infection time (d = 0.987). These results indicate that the established method for quantitative detection of C. elegans nuclear polyhedrosis virus by real-time fluorescence quantitative PCR is reliable, has high sensitivity and good repeatability, and can be used for the dynamic study of in vivo proliferation of insect nuclear polyhedrosis virus and the standardization of insect viruses Production for reference.