转基因白桦外源基因的多重PCR快速检测

A multiplex polymerase chain reaction method for rapid detection of foreign genes in transgenic birch (Betula platyphylla)

根据转化的载体序列上T-DNA中的目的基因bt,选择性筛选标记基因nptⅡ和报告基因gus设计三对特异性引物,PCR产物片断大小分别为247bp、449bp、668bp,应用多重PCR(muti-plex-PCR)的方法同步检测18株转基因白桦中三个基因的整合状况;用阳性对照为模板,对单重PCR(simplex-PCR)和多重PCR的各项指标进行比较。结果表明多重PCR检测多个外源基因在敏感性方面与单重PCR相比并没有减弱,而且略有提高;对18株样品的多重PCR同步检测无假阳性出现,结果准确,同时在操作中具有减少污染,缩短时间和节约成本等优点。因此,在对转基因白桦的外源基因的定期检测中,多重PCR是一种非常有效而便捷的方法,可以为转基因的拷贝数,T-DNA旁侧序列特征等转基因整合特性方面的研究提供数据。

A multiplex polymerase chain reaction （mPCR） assay was developed to simultaneously detect target gene （bt）, seleetable marker gene （nptⅡ） and reporter gene （gus） of T-DNA from 18 transgenie birch. Three sets primers were designed and synthesized based on transferred vector sequence. Under the optimized conditions, the assay yielded a 247 bp DNA fragment from bt gene, 449 bp DNA fragment from nptⅡ gene, 668 bp DNA fragment from gus gene. The results of the mPCR using positive control showed that the sensitive and simultaneous detection of foreign genes were as normal as simplex PCR assay, also a little higher. After simultaneous detecting 18 transgenie birches using mPCR, we found that the method could decrease the risk of contamination, save time and reduce the cost and so on. The mPCR method provides a useful rapid technique for detecting multiple genes in transgenie birch and offers data on transgenie copy number, flanking sequences of transgenie T-DNA, and some other transgenie integration researches.