实时LAMP法快速检测食用植物油中的转基因成分CaMV-35S

Rapid Detection of CaMV-35S Promoter in Vegetable Oils by Loop- mediated Isothermal Amplification Method

环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)是一项新的DNA扩增技术,近年来已被成功应用于转基因作物中外源基因的检测分析。本文针对花椰菜花叶病毒35 S启动子(CaMV-35S)设计引物以及有效提取目标DNA后,首次建立了食用植物油中CaMV-35S启动子的LAMP检测方法。通过在DNA提取过程中加入正己烷乳化和加入共沉淀剂,获得了可以进行LAMP扩增的DNA片段,采用LAMP实时浊度法和染色法对扩增产物进行比较分析。着重对FIP/BIP引物、甜菜碱和Mg2+浓度等反应参数进行了优化。结果表明,LAMP方法能够在56 min内特异性地检测到CaMV-35S启动子,检测灵敏度比常规PCR高10倍,而且不需要特别的仪器设备,扩增产物可通过观察实时浊度曲线或通过SYBR Green I染色后借助肉眼对检测结果进行判断。该方法快速高效、操作简便、灵敏度高、检测结果准确,适合食用植物油中转基因成分的快速检测。

The Loop-mediated isothermal amplification （LAMP） assay is a novel and altemative DNA amplification method, which amplifies DNA with high specificity and efficiency, and has been successfully used to screen genetically modified （GM） crops （e.g., soybean, maize）. In the present study, a LAMP assay for the detection of exogenous cauliflower mosaic virus 35 S promoter （CaMV-35S） in vegetable oils was firstly developed. The genomic DNA of vegetable oils was effectively extracted by adding hexane and co-precipitant. In the LAMP assay, and the reactions were optimized, involving different concentrations of FIP/BIP primer, betaine and Mg2＋. Two different platforms of measurement （real-time turbidity and fluorescence dyes） were compared. Results indicated that LAMP method was rapid （within 56 min） and sensitive, and the detection sensitivity was ten fold higher than that of the conventional PCR. Without specialized PCR or electrophoresis instruments, the target DNA was amplified under isothermal conditions （63 ℃） and visualized by accumulated curve of turbidity or staining inspection （SYBR Green I） for naked-eye. The CaMV-35S promoter was successfully detected in vegetable oil samples （soybean oils and rapeseed oils） by this method. The LAMP approach was rapid, sensitive, and available for the monitoring of transgenic elements in GM vegetable oils.