5种转基因油菜转化体特异性多重PCR检测方法

【目的】全球转基因植物及其产品的数量和种类越来越多,迫切需要可同时精准高效检测多个转化载体的检测方法。【方法】针对RF1、MS8、Topas19/2、Oxy235和RF3等5个转基因油菜品系的侧翼序列及油菜内源基因cruciferin A(Cru A)序列设计多重聚合酶链式反应特异性引物,通过对转基因油菜、转基因大豆、转基因玉米、转基因水稻、转基因棉花等不同作物进行PCR扩增来测试所选择的引物特异性,优化多重PCR反应引物的浓度,用所建立的检测体系对不同混合比例的转基因油菜进行多重PCR扩增来测试所建立的检测方法的灵敏度。【结果】通过测试,仅在含有目标样品中检测出阳性结果,灵敏度达0.05%,表明所建立的6重PCR检测方法可同时精准检测RF1、MS8、Topas19/2、Oxy235和RF3等5种转基因油菜转化载体。【结论】所建立的6重转基因油菜转化体特异性PCR检测方法通量高、特异性好、灵敏度高,符合有关转基因产品检测的要求,可作为转基因油菜检测的有效方法。

Event-specific multiplex PCR detection method for five genetically modified canola lines

Aim] With the increased number and variety of genetically modified (GM) plants and their derived products in the world, detection methods that can efficiently detect multiplex transformation vector simultaneously become increasingly important.Method] Based on flanking sequences of five GM canola line (RF1, MS8, Topas19/2, Oxy235 and RF3) and endogenous reference gene cruciferinA (CruA) for canola, event-specific primers were designed. To analyze specificity and sensitivity of primers, GM canola lines, GM soybean lines, GM maize lines, GM rice lines and GM cotton lines were amplified by single PCR and different DNA concentrations of GM canola were amplified by multiplex PCR.Result] The developed 6×multiplex PCR assay could specifically detect five canola lines, RF1, MS8, Topas19/2, Oxy235 and RF3. The sensitivity of multiplex PCR detection was 0.05%.Conclusion] The developed 6×multiplex PCR system has high efficiency, specificity and sensitivity, and could meet the requirements of transgenic component detection. All these results suggested the developed multiplex PCR system could be applied in GM canola detection.