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基于TaqMan实时荧光定量PCR技术的西花蓟马快速检测
TaqMan realtime fluorescent quantitative PCR for identification of Frankliniella occidentalis
吴霞1**,张桂芬1, 2 ***,万方浩1, 2***
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DOI:
作者单位:1中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室北京100193;2 农业部外来入侵生物预防与控制研究中心北京100081
中文关键词:西花蓟马,TaqMan-MGB探针,实时荧光定量PCR,快速检测,SCAR标记
英文关键词:Frankliniella occidentalis, TaqMan-MGB, realtime fluorescent quantitative PCR, rapid detection, SCAR marker
中文摘要:
      西花蓟马Frankliniella occidentalis (Pargande)是世界性害虫,2003年在我国首次发生危害。针对西花蓟马与其他种类蓟马形态相似、难以快速区分的问题,本文在SCAR标记基础上,采用TaqMan实时荧光定量PCR技术,设计1对特异性引物和1条MGB探针,扩增出大小为138 bp的特异片段。以质粒DNA为标准品建立了标准曲线(R2 = 0.9965),种特异性检验结果显示,该引物和探针只能检测到西花蓟马的荧光信号,而对其他种类的蓟马不具有检测能力。并且可以定量检测西花蓟马不同虫态靶标DNA片段的拷贝数。该检测体系重复性强、稳定性高,在口岸检疫以及植物种苗及其产品调运中的有害生物检测和监测中具有重要意义。
英文摘要:
       The western flower thrip, Frankliniella occidentalis (Pargande), an invasive species worldwide, was first reported in Beijing in 2003 Morphological identification of thrips is difficult because of their small size, high degree of similarity, and polymorphism. In contrast, molecular identification is not hindered by phenotypic polymorphism, or the sex or stage of development of the target species. We evaluated the effectiveness of TaqMan realtime fluorescent quantitative PCR to identify WFT, for which we developed a pair of species specific primers and a MGB probe. The amplified fragment size was 138 bp. Standard points made using serial dilutions of plasmid DNA had a strong linear relationship with the detected Ct values (R2 = 0.9965), making the Ct value a reliable way to quantify DNA. Specificity tests performed with the TaqMan-MGB probe and primer sets showed that all F. occidentalis specimens were detected and that there were no cross reactions with 8 other thrip species, including F. intonsa (Trybom) and F. tenuicornis (Uzel). Moreover, the system could quantitatively detect target DNA copies from all developmental stages of F. occidentalis with high repeatability and stability The results indicate that our detection system has high sensitivity and specificity and can be used for on-site testing of samples at portsofentry and during transportation of plant seedlings and products.
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