| 封立平1, 李洪林2, 倪新3, 王简1, 纪瑛1, 甘琴华1.木薯细菌性萎蔫病菌的LAMP 快速检测方法[J].植物保护,2015,41(3):93-99. |
| 木薯细菌性萎蔫病菌的LAMP 快速检测方法 |
| Loop-mediated isothermal amplification (LAMP) for rapid detection of Xanthomonas axonopodis pv. manihotis |
| 投稿时间：2014-12-17 修订日期：2015-03-09 |
| DOI： |
| 中文关键词: 木薯细菌性萎蔫病菌 环介导等温扩增 检测 |
| 英文关键词:Xanthomonas axonopodis pv. manihotis LAMP detection |
| 基金项目:国家质检总局科技计划项目(2009IK254; 2011IK286); “十二五”国家科技支撑计划(2012BAK11B02); 山东出入境检验检疫局科研项目(2005SK09) |
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| 中文摘要: |
| 建立了一种木薯细菌性萎蔫病菌的环介导恒温扩增快速检测方法, 为木薯细菌性萎蔫病的快速检测提供有力的技术支持。针对木薯细菌性萎蔫病菌TAL效应器蛋白质(pthBXam)靶序列的6个位点设计4条特异性引物, 并对反应温度和内引物浓度等参数进行了优化, 设计的引物与试验中提供的其他黄单胞近缘种都没有扩增反应, 表现了较好的特异性。LAMP 方法对木薯细菌性萎蔫病菌菌株DNA的检测下限为1 pg/μL, 比常规PCR灵敏度高100倍。该方法采用SYBR Green I染料法对扩增产物闭管检测, 裸眼观察颜色变化判断反应结果, 能快速、准确地对田间样品进行检测, 没有出现假阳性和假阴性。与其他检测方法相比, LAMP 方法检测时间短, 效率高, 降低了设备投入, 易于操作, 适合木薯细菌性萎蔫病菌的现场检疫和大规模监测。 |
| 英文摘要: |
| A rapid loop-mediated isothermal amplification (LAMP) method was established for quick detection of Xanthomonas axonopodis pv. manihotis. The target sequence pthBXam was amplified by a set of four specific primers that recognize six distinct target sequences. The parameters, including the reaction temperature and the inner primer concentrations, were optimized. The LAMP primers did not react with other closely related Xanthomonas in this test. The lower limit of detection of this LAMP assay was about 1 pg/μL DNA, which was 100 times more sensitive than PCR method. The amplification products could be checked by naked-eye visualization by a closed-tube detection technique with SYBR Green I staining. The LAMP method is simple and rapid method for detection of X. axonopodis pv. manihotis from the infected cassava sampled in the field. The LAMP assay has advantage of short detection time, high efficiency and low cost, which provides a new tool for on-site quarantining and large-scale monitoring of this pathogen. |
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