转基因番木瓜“华农一号”事件特异性定性PCR检测方法的建立

以我国自主研发的转基因抗环斑病毒番木瓜"华农一号"为研究对象,应用TAIL-PCR技术,用载体中靠近右边界的Nos启动子序列设计特异引物,扩增插入的旁邻序列;根据测序结果,设计了产物长度分别为753和298 bp但均跨越转化载体和番木瓜基因组序列的2套特异引物,对"华农一号"进行检测,证明其可特异地把"华农一号"从转基因番木瓜品系中检测出来;2对引物的检测灵敏度都可以达到0.05%的标准,高于欧盟0.9%的检测要求;利用2对特异引物对"华农一号"的果肉、果皮、种子进行检测,可得到特异性良好的扩增.

Establishment of Event-Specific Qualitative PCR Detection Method for Transgenic Papaya "Huanong No.1"

Event-specific qualitative PCR detection method has advantageous because of its specificity and hence be used in the Chinese detection standards.In this study,an event-specific transgenic detection method for 'Huanong No.1',a transgenic papaya variety with high resistance to papaya ringspot virus,and widely planted in Guangdong Province,was established.By using the thermal asymmetric interlaced PCR(TAIL-PCR) technique,the flanking sequences of the exogenous integration in the genome of 'Huanong No.1' were cloned and sequenced with the specific nested primers based on Nos promoter near the right border(RB).According to these sequences,two pairs of PCR primers were designed to amplify the fragments with sizes of 753 and 298 bp respectively,and spanning the exogenous integration and papaya genome.Hence,the qualitative PCR assays were established for 'Huanong No.1'.The detection sensitivity reached to 0.05%.Furthermore,this method can also be used in various organs of papaya,such as seeds,fruits,fruit skins etc.