双重PCR-毛细管电泳法快速检测大豆中转基因成分

Rapid Analysis of Genetically Modified Soybean by a Duplex PCR-capillary Electrophoresis System with Laser-induced Fluorescence Detection

目的 建立抗草甘膦转基因大豆的快速检测方法。方法 针对转基因大豆基因组中被导入的 35 S启动子、NOS终止子和 CP4 - EPSPS抗草甘膦基因等外源基因 ,自行设计了两对引物 ,采用双重 PCR同时扩增上述基因 ,用 8g/L羟丙基甲基纤维素为筛分介质 ,在 5 0 cm× 10 0μm i.d.涂壁毛细管中 ,于 - 10 k V电压下用激光诱导荧光 -毛细管电泳检测转基因大豆的 PCR扩增产物。结果 在优化的 PCR反应和毛细管电泳条件下 ,本法可以同时检测出转基因大豆样品中三种外源基因 ,双重 PCR扩增产物经测序证实与原基因序列完全一致 ,表明本研究设计的引物合理 ,扩增结果可靠。毛细管电泳进样量仅需 5 nl,分析时间为 2 4 min,迁移时间的相对标准偏差≤ 3.2 %。结论 本方法较常规琼脂糖凝胶电泳特异性高 ,分析时间短 ,重现性好 ,检测灵敏 。

Objective To develop a rapid detection method for genetically modified soybean resistant to glyphosate. Methods A duplex PCR was performed with primers designed in this study to simultaneously amplify heterogenous genes in transgenic soybean:CaMV-35S promoter, NOS terminator and CP4-EPSPS gene. And a simple capillary electrophoresis with laser-induced fluorescence detection(CE-LIF) was developed and applied to the rapid analysis of the above PCR products by using a 50 cm length ×100 μm i.d. capillary coated with linear polyacrylamide and an 8 g/L HPMC-4000 sieving buffer under 200 V/cm electric field strength. Results The proposed method was able to simultaneously detect the three heterogenous genes existing in genetically modified soybean under the optimization conditions of PCR and capillary electrophoresis. The measured sequences of the duplex PCR products were identical with the original genes' sequences. Moreover, the sample volumes required were not more than 5 nl and the detection could be completed in less than 24 min. The relative standard deviations (R.S.D) of the migration times for the PCR products were≤3.2%. Conclusion In comparison with agarose gels electrophoresis, the duplex PCR-based capillary electrophoretic method with laser-induced fluorescence detection is rapid, sensitive and accurate, and it is suitable for detection of genetically modified soybean.