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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 classified as a quarantine pest of entry phytoplankton by our country, D. virgifera virgifera, D.undecimpunctata, D. barberi is a major agricultural pest in North America. In order to establish a rapid molecular identification method to identify these three kinds of Diabrotica, the present study obtained some mtDNA COI sequences from adult samples collected from the field and compared with the relevant sequences in Gen Bank to design and screen 3 Specific primers and TaqMan probes were used to detect the specificity and sensitivity of real-time fluorescent PCR. The results of the specificity test showed that near-S-type amplification curves appeared in 30 cycles (Ct value <30) when the real-time fluorescent PCR reaction of the target species Diabrotica DNA and its specific probes and primers, At the same time, no other species of fluorescence signal growth. In addition, the sensitivity results showed that the minimum DNA template concentration detectable by A. lucidum and P. polygamis, 0.1 ng / μL for real-time PCR, 0.01 ng / μL for O. elegans, μL.