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当前位置: 首页 > 文章 > 番茄溃疡病菌LAMP快速检测方法的建立 河北农业大学学报 2015 (3) 19-23
Position: Home > Articles > Development of a loop-mediated isothermal amplification assay for detection of Clavibactermichiganensissubsp.michiganensis Journal of Agricultural University of Hebei 2015 (3) 19-23

番茄溃疡病菌LAMP快速检测方法的建立

作  者：
赵赛;李建嫄;周颖;杨文香;贵亚欣;张娜;刘大群
单  位：
河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术研究中心/国家北方山区农业工程技术研究中心
关键词：
番茄溃疡病菌;环介导等温扩增方法;快速检测
摘  要：
以SYBR Green I为指示剂,建立了番茄溃疡病菌的环介导等温扩增可视化快速检测方法。根据番茄溃疡病菌特异的核糖体转录间隔区序列设计了4条引物进行LAMP扩增,通过对体系中各成分进行优化,最终确定25μL反应体系中模板200ng,MgSO4的适宜浓度为3.0mmol/L,Bst DNA聚合酶2U,dNTPs的适宜浓度为0.3mmol/L,外引物与内引物之比为1∶3时扩增效果较好,在65℃最佳反应时间为40min。在优化的体系条件下,获得的反应产物经SYBR Green I染色后,肉眼观察检测灵敏度可达到50CFU/mL,且对番茄溃疡病菌的检测具有高度的特异性。结果表明,该方法快速、准确、灵敏、特异,具有良好的实用性。
译  名：
Development of a loop-mediated isothermal amplification assay for detection of Clavibactermichiganensissubsp.michiganensis
作  者：
ZHAO Sai;LI Jian-yuan;ZHOU Ying;YANG Wen-xiang;GUI Ya-xin;ZHANG Na;LIU Da-qun;College of Plant Protection,Agricultural University of Hebei/Biological Control Center of Plant Diseases and Plant Pests of Hebei Province,China/National Engineering Research Center for Agriculture in Northern Mountainous Areas;
关键词：
Clavibacter michiganensis subsp.michiganensis;;loop-mediated isothermal amplification;;rapid detection
摘  要：
A visual loop-mediated isothermal amplification(LAMP)detection of bacterial canker and wilt of tomato was developed with SYBR Green I as a fluorescence indicator in this study.Based on the special internal transcribed spacer(ITS)sequence of Cmm,a set of four LAMP primers was designed.The final reaction system is:template DNA 200 ng,the optimum concentration of MgSO4 was 3.0mmol/L,Bst DNA polymerase 2U,optimal concentration of dNTPs for 0.3mmol/L,and the ratio of outer primers and internal primers was 1∶3for optimum amplification in the total 25μL reaction system.The results showed that the constant temperature of 65 ℃for 40 min could acquire good amplification results.The detection sensitivity of LAMP was 50CFU/mL by naked eyes detection after SYBR Green I dying,theprimers were high specific for Cmm detecting.The LAMP assay developed in this study was simple,fast,sensitive and specific,with good practicability.

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