抗草甘膦转基因大豆Cp4-epsps基因快速简便的LAMP检测方法  

Rapid and simple detection of Cp4-epsps gene in glyphosate-resistant soybean by loop-mediated isothermal amplification

作　　者：朱琳峰 彭焕[1] 黄文坤[1] 张东升[1] 孔令安[1] 彭德良[1] 

机构地区：[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

出　　处：《植物保护》2015年第3期86-92,共7页Plant Protection

基　　金：转基因生物新品种培育科技重大专项(2014ZX008011-003)

摘　　要：针对抗草甘膦转基因大豆的外源基因Cp4-epsps,建立了一种基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术的抗草甘膦转基因大豆的检测体系,其扩增产物既可利用常规琼脂糖凝胶电泳检测,还可通过SYBR Green I染色进行快速检测。LAMP检测体系中dNTPs浓度为0.8 mmol/L、Mg2+浓度为3mmol/L、反应时间为45min时扩增效果最佳,其检测灵敏度为5μg/L,比常规PCR灵敏100倍。田间实际检测结果表明,LAMP检测结果和PCR检测结果完全一致,准确率为100%。本研究所建立的抗草甘膦转基因大豆LAMP检测方法具有简便快速、特异性强、灵敏度高等特征,是一种能够用于抗草甘膦转基因大豆检测、田间基因漂移监测和环境安全研究的有力工具。The LAMP （loop-mediated isothermal amplification） assay based glyphosate-resistant soybean detection system was developed in this paper. A set of four basic primers and two loop primers was designed to target the ex- ogenous Cp4-epsps gene. The LAMP products were detected by agarose gel electrophoresis and SYBR Green l. The optimum result was obtained at 45 rain of reaction time, 0.8 mmol/L dNTPs and 3 mmol/L Mg^2＋ . The meth- od developed in this paper could specifically detect glyphosate-resistant soybeans. And the low detect limit of ge- nomic DNA of glyphosate-resistant soybeans was 5 μg/L which was 100 times higher than that of regular PCR. The field testing showed that the results of LAMP detection were consistent with those of PCR, and the accuracy of this method was 100%. The LAMP method we developed for glyphosate-resistant soybean detection is rapid,ef- ficient, specific, sensitive and convenient, which make it a useful tool for detection of glyphosate-resistant soybean, monitoring of gene flow and research on environmental security.

关 键 词：抗草甘膦转基因大豆 Cp4-epsps LAMP 快速检测 

分 类 号：S565.1[农业科学—作物学]