作　　者： ; ; ; ; ; (刘唐书）;

机构地区： 南方医科大学基础医学院基因工程研究所

出　　处： 《中国食品学报》 2013年第4期149-155,共7页

摘　　要： 目的:建立一种快速检测CaMV35S启动子基因的环介导等温扩增技术检测方法。方法:针对CaMV35S启动子设计4对特异性引物,在链置换酶(Bst酶)作用下,65℃扩增1h。通过对转基因大豆GTS40-3-2等8种转基因标准品的检测,对该方法进行特异性评价。以F3、B3为引物,转基因大豆GTS40-3-2为模板,纯化后的PCR扩增产物与pMD-18T载体连接,构建含有CaMV35s启动子的质粒标准分子。配制7个浓度梯度的标准质粒分子,进行灵敏度分析。同时应用该方法和荧光PCR方法检测35种农产品及其加工品。结果:该方法特异性强,结果可视,检测灵敏度达200copies/μL,35种样品的检测结果与SYBRGreen荧光PCR检测结果一致。结论:建立的LAMP法具有快速,简单,可视化,灵敏度高和特异性强等优点,可应用于转基因产品的初步筛选。 Objective： To establish a LAMP assay for rapid detection the 35S promoter of cauliflower mosaic virus. Methods： A set of four specific primers were designed to target the CaMV35S gene. The amplification could be obtained in 60 min by incubating all of the reagents in a single tube with Bst DNA polymerase at 65 ℃. 8 kinds of genetically modified（GM） standards were applieded to evaluate the specificity of the LAMP. A standard plasmid molecules was con- structed to make dilutions for evaluation of the lower detection limit. Under optimized conditions, the LAMP method and fluorescence. PCR were performed to detect the CaMV35S gene in 35 kinds of samples simultaneously. Results： The LAMP method is highly specific, its amplification products can be detected by direct eye inspection and the detection sensitivity was up to 200 copies/μL. The analyzed results of 35 kinds samples by LAMP were in good consistent with the results by fluorescence PCR. Conclusion： This LAMP method was rapid, visualized, highly sensitive and specific, it can be widely used in initial screening for GM products.

关 键 词： 花椰菜花叶病毒 环介导等温扩增技术 转基因食品

领　　域： [轻工技术与工程] [轻工技术与工程]