摘要
根萤叶甲属(Diabrotica)被我国列为进境植物检疫性有害生物,该属中的玉米根萤叶甲(D.virgifera virgifera)、十一星根萤叶甲(D.undecimpunctata)、巴氏根萤叶甲(D.barberi)是北美的主要农业害虫。为了建立一种快速的分子鉴定方法以鉴定这3种根萤叶甲,本研究从田间采集的成虫样品中获得其部分mtDNA COI序列,与Gen Bank中的相关序列进行比对,设计筛选出3种根萤叶甲的特异性引物和TaqMan探针,并进行实时荧光PCR特异性和灵敏度检测。特异性检测结果表明,用目标种根萤叶甲DNA和其特异性探针和引物进行实时荧光PCR反应时,在30个循环反应内(Ct值<30)有近S型扩增曲线出现,同时其他种均无荧光信号增长。此外,灵敏度结果显示,玉米根萤叶甲和巴氏根萤叶甲可检测的最小DNA模板浓度,即实时荧光PCR反应的灵敏度为0.1 ng/μL,十一星根萤叶甲为0.01 ng/μL。
Diabrotica is listed as the quarantine pest of entry by China. Among the genus,D. virgifera virgifera,D. undecimpunctata and D. barberi are major agricultural pests in North America. To develop a rapid method of molecular identification of these Diabrotica species,partial sequences of mitochondrial DNA cytochrome oxidase submit I gene（mtDNA COI） were obtained from the field-collected specimen,and were compared with the corresponding sequences in the Gen Bank. The species-specific primers and TaqMan probes were designed and tested with six concentrations of template DNA in real-time PCR.With matched template DNA and primers/probe,the exponential phase in the real-time PCR occurred at the threshold cycles smaller than 30（Ct values 〈 30） while no such curve showed up for any unmatched DNA and primers among the three species. Furthermore,the detectable template DNA concentration,as the sensitivity of the real-time PCR method developed in this study,was as lower as 0.1 ng/μL for both D. virgifera virgifera and D. barberi,and went ever lower to 0.01 ng/μL for D. undecimpunctata.
出处
《植物检疫》
北大核心
2017年第5期38-43,共6页
Plant Quarantine
基金
国家科技支撑计划项目课题(2012BAK11B01)
