TaqMan实时荧光定量PCR检测美国白蛾核型多角体病毒方法的建立

Development of the TaqMan fluorescent quantitative PCR for detection of Hyphantria cunea nudeopolyhedroviurs

美国白蛾核型多角体病毒( Hyphantria cunea nudeopolyhedroviurs)是一种对美国白蛾高效、对环境无污染、对天敌及非靶标生物安全的生物杀虫剂。建立高效、快速的定量检测方法对该病毒持续控制美国白蛾具有重要意义。将 polh 基因构建到pGEM-T-easy载体上,制备标准品质粒,构建标准曲线,对未知样品DNA进行定量PCR检测,根据标准曲线计算样品中美国白蛾核型多角体病毒含量。提取感染病毒不同时间段的美国白蛾幼虫基因组DNA,根据已建立的方法检测并计算每克样本病毒的基因拷贝数。利用TaqMan探针法成功建立了该病毒荧光定量PCR检测方法。以取食3.0×10^6 OBs/mL浓度病毒的美国白蛾幼虫基因组DNA为模版,进行荧光定量PCR,病毒在幼虫体内的含量在第5天开始出现大幅增长趋势,第6天时达到最高。TaqMan探针法实时荧光定量PCR能简便、高效、快速的检测出大量未知样本基因组DNA中病毒基因的拷贝数,为研究该病毒的传播和流行规律提供了可行的监测方法。

Hyphantria cunea nudeopolyhedroviurs is a biological pesticide with the highly efficiency to its host, pollution-free to environment and safety to natural enemies and non-target organisms. Establishing an efficient and rapid method for the quantitative detection of the virus has a great significance for the sustainable control against H.cunea. In this paper, we constructed the polyhedrin gene into the pGEM-T-easy vector and prepared the standard plasmid and constructed the standard curves. and then the DNA of unknown sample was detected by quantitative PCR. According to the standard curve, we calculated the content of nudeopolyhedroviurs in the sample. Extracted the genomic DNA of the larva at different times after infection, and then detected the copy number of the virus gene per mg sample, based on the established method. We established a detection method of fluorescent quantitative PCR using the TaqMan probe. Based on the genomic DNA template of the larvae infected 3.0×10^6 OBs/mL virus, we performed the fluorescence quantitative PCR and found that the content of virus had a significant upward trend at 5 d on the larvae and reached a maximum at 6 d. The real-time fluorescence quantitative PCR with TaqMan probe could detect the copy number of virus on the unknown sample DNA with quickly and efficiently. It provides a feasible method for monitoring the transmission and prevalence of the virus.