通用引物多重PCR新技术对番木瓜转基因成分检测的研究

Detection of Genetically Modified Ingredients in Transgenic Papaya using Universal Primer-multiplex PCR

为了满足能够同时对多个靶标进行检测,克服传统多重PCR的引物间排斥、重现性差等缺点的要求,建立通用引物多重PCR新方法对番木瓜中转基因成分进行高效、快速的检测。选用三种转基因番木瓜(Event 55-1、"GM-YK"和"华农一号")和非转基因番木瓜("台农2号")的新鲜叶片为研究对象,根据三种转基因番木瓜的外源基因和内标木瓜蛋白酶基因(papain)序列,设计五种特异性引物。通过比较不加入接头的特异性引物的PCR验证结果,发现复合引物(带通用接头的引物)无论在单重PCR或者多重PCR的检测中的电泳条带均更为明亮清晰。而由灵敏度的对比,可以得出通用引物多重PCR灵敏度比普通引物的灵敏度提高了1个数量级。通用引物多重PCR新技术较普通多重PCR方法检测番木瓜中转基因成分拥有更低的检测限和灵敏度,为转基因成分的快速检测提供一种新技术。

In order to meet the need for simultaneous detection of multiple targets and to overcome the several limitations associated with traditional multiplex polymerase chain reaction（PCR） such as primer exclusion,poor reproducibility etc.,a novel universal primer-multiplex PCR approach was developed to efficiently and rapidly detect the genetically modified ingredients in papaya.Fresh papaya leaves from three transgenic papaya cultivars（Event 55-1,＂GM-YK＂,＂Huanong No.1＂） and non-transgenic papaya（＂Tainong No.2＂） were chosen for this study.Five specific primers were designed based on the sequences of the exogenous genes of the three transgenic papaya cultivars and endogenous reference gene（papaya proteinase）.The electrophoretic bands of the composite primers（with universal adapter primers） were brighter and clearer in both simplex PCR and multiplex PCR detections,compared to the PCR results of primers without the addition of the adapters.The sensitivity of multiplex PCR using universal primers was one order of magnitude higher than that detected using common primers.Compared to ordinary multiplex PCR,the novel universal primer-multiplex PCR has a lower detection limit and higher sensitivity in the detection of genetically modified ingredients in transgenic papaya,providing a new technique for the rapid detection of genetically modified ingredients.