转基因玉米的多重PCR－毛细管电泳紫外检测技术研究

Study on genetically modified maize by multiplex polymerase chain reactioncapillary electrophoresis with UV detection

建立了转基因玉米Ly038、Mon863和Mon810的品系特异性基因多重PCR产物的毛细管电泳-紫外检测方法。根据三种转基因玉米的基因序列设计多重PCR引物,优化PCR扩增体系和条件,以8.0g/L羟乙基纤维素为筛分介质,用毛细管电泳-紫外检测法同时检测出三种玉米的品系特异性基因,11.195min内即可完成检测。用Origin软件对电泳结果进行分析并得出不同范围的DNA曲线方程,样品出峰时间的相对误差在1.03%～5.02%之间。并对纯化前后的样品进行了毛细管电泳分离的对照,发现PCR产物纯化后更适于紫外检测的毛细管电泳。多重PCR具有节约模板、节省试剂和缩短检测时间的优点,毛细管相对于传统的琼脂糖凝胶电泳,具有高效、快速、灵敏且经济环保的优点,所以此方法可广泛地应用于食品安全检测和临床检验等领域中。

Event-specific qualitative detection of three kinds of genetically modified（GM）maize by multiplex polymerase chain reaction-capillary electrophoresis（CE）with UV detection was established. Three sets of primers were designed to amplify the event-specific fragments of GM maize events（Ly038,Mon863 and Mon810）. Multiplex PCR and the separation of CE were systematically optimized,8.0g/L hydroxyethylcellulose in tris-phosphate-EDTA（TBE）buffer as sieving matrix,three event-specific multiplex PCR products were detected by CE in 11.195min.Using Origin program to analyze the CE results and also get the fitting curves of different DNA range,the relative error was between 1.03% and 5.02%. Unpurified and purified normal PCR products were also detected by CE which suggested purification of PCR products was important for CE with UV detection. Compared with normal PCR and agarose gel electrophoresis,the multiplex polymerase chain reaction-capillary electrophoresis method in this study was efficient,rapid,sensitive and low-cost. This event-specific detection method could be widely applied in the fields of food safety and clinical tests.