转基因作物外源转基因成分环介导等温扩增技术检测方法的建立及应用

 【目的】建立转基因作物外源转基因成分环介导等温扩增技术检测方法。【方法】以转基因作物中常用的花椰菜花叶病毒35S启动子（CaMV35S）为主要研究对象，利用环介导等温扩增技术（loop-mediated isothermal amplification，LAMP），针对CaMV35S基因的6个区域设计4种特异引物，利用1种链置换DNA聚合酶(Bst DNA polymerase)在65℃保温1 h，通过荧光显色即可完成对转基因作物鉴定工作。同时对7种转基因作物进行LAMP检测。【结果】该LAMP方法通过特异性检测含有CaMV35S启动子的转基因作物，其检测结果的特异性与常规PCR方法结果一致，灵敏度是常规PCR的10倍。【结论】转基因作物LAMP检测方法具有高度的特异性及稳定性，结果可靠，非常适合转基因作物的快速检测，有较好的应用价值。

Development and Application of Loop-Mediated Isothermal Amplification for Detection of Genetically Modified Crops

【Objective】 The objective of the study is to develop a loop-mediated isothermal amplification method for detection of genetically modified crops(GMC). 【Method】 The detection method is based on the loop-mediated isothermal amplification (LAMP) reaction. Cauliflower mosaic virus 35S (CaMV35S) promoter gene, a widespread genetic element, was amplified by a set of four specially primers that recognize six distinct sequences of the target. The amplification can be obtained in 1 h by incubating all of the reagents in a single tube with Bst DNA polymerase at 65℃. 【Result】 The LAMP method can detect a specific promoter containing CaMV35S from GMC and their test results were consistent with the results of conventional PCR methods. LAMP assay results were found to be 10 times more sensitive than the conventional PCR. 【Conclusion】 The LAMP detection method is specific, stable and reliable, and will be an effective tool for rapid detection of GMC.