转cry1Ab/c水稻环介导等温扩增快速检测方法  

Rapid Detection of cry1Ab/c Gene in Transgenic Insect-Resistant Rice Using Loop-Mediated Isothermal Amplification Method

作　　者：王镓璇 胡昱萱 吴海星 檀建新[1] WANG Jiaxuan;HU Yuxuan;WU Haixing;TAN Jianxin(College of Food Science and Technology,Hebei Agricultural University,Baoding 071001)

机构地区：[1]河北农业大学食品科技学院,保定071001

出　　处：《食品工业》2020年第5期215-219,共5页The Food Industry

基　　金：河北省质量工程(1009031);大学生创新创业训练计划(0321002);河北省食品科学与工程学科“双一流”建设资金项目(2016SPGCA18);河北省重点研发计划项目(16275505D)。

摘　　要：为快速检测水稻中是否含有crylAb/c抗虫基因,利用LAMP在线引物设计软件针对目的基因的6个区域设计筛选4条特异性引物,利用Bst DNA聚合酶在65℃条件下恒温扩增1 h,通过肉眼观察浑浊度、凝胶电泳法和荧光染料法三种方法来显示检测结果,并对LAMP反应体系中各影响因素进行优化,并确定LAMP最佳反应体系。通过对LAMP方法与PCR方法检出限的比较,证明其检出限比PCR方法低1/10。同时用2种转基因作物和4种非转基因作物检测LAMP方法的特异性,结果显示其特异性与PCR检测一致。说明LAMP方法检出限低、特异性好,适用于转基因水稻中crylAb/c基因的检测。In order to quickly detect whether rice contains cry1 Ab/c insect-resistant genes, this study used loop-mediated isothermal amplification(LAMP) online primer design software to screen 4 specific primers for 6 regions of the target gene. After amplifying under an isothermal temperature of 65 ℃ for 1 hour using Bst DNA polymerase, the results were checked through checking the turbidity by the naked eye, the DNA pattern on gel electrophoresis and signal of fluorescent dye. The optimum reaction system of LAMP was determined by optimizing the influencing factors in the LAMP reaction system. By comparing the detection limit of LAMP method with that of PCR method, it is proved that the detection limit is one-tenth lower than that of PCR method. At the same time, 2 transgenic crops and 4 non-transgenic crops were used to detect the specificity of the LAMP method, which showed that the specific results were consistent with that of PCR test. It shows that LAMP method has lower limit and high specificity, and it is suitable for the detection of cry1 Ab/c gene in transgenic rice.

关 键 词：转基因抗虫水稻 环介导等温扩增 检测 cry1Ab/c Bst DNA聚合酶 

分 类 号：S511[农业科学—作物学] TS210.1[轻工技术与工程—粮食、油脂及植物蛋白工程]