利用线粒体coxⅡ内含子对西洋参和人参的分子鉴别

Identification of Panax ginseng and Panax quinquefolius by Mitochondrial Cox Ⅱ Intron

目的:基于西洋参和人参在线粒体细胞色素C氧化酶亚基Ⅱ(coxⅡ)的内含子对西洋参和人参进行鉴别,为人参类药材提供简单准确的分子检测手段。方法:利用通用引物对人参和西洋参线粒体coxⅡ基因的内含子进行聚合酶链式反应(PCR)扩增,将扩增后的序列进行双向测序,通过序列比对挖掘人参和西洋参的插入/缺失序列。根据人参和西洋参的特异性插入序列分别设计两者的品种特异性引物,建立鉴别人参和西洋参的多重PCR体系,并确定该多重PCR体系的检测限。结果:在线粒体coxⅡ的内含子中发掘了人参和西洋参的插入/缺失序列。在多重PCR体系下,人参产生了729 bp的条带,西洋参产生了141 bp的条带,人参和西洋参的混合品则分别产生了729 bp和141 bp的条带。该多重PCR体系可以检测人参中0.1%的西洋参掺入,检测限低至0.001 ng。结论:该方法建立的多重PCR体系既不需要对反应体系进行优化,也不需要引入额外的碱基错配,可以对不同来源的人参和西洋参进行准确的鉴定,为人参和西洋参植物来源的鉴定提供了新的分子标记方法。

Objective:To develop a simple and accurate method for molecular authentication of Panax ginseng and P. quinquefolius. Method: The mitochondrial cox Ⅱ sequences of P. ginseng and P. quinquefolius were amplified by polymerase chain reaction(PCR)with universal primers. PCR products of the two species were sequenced in both directions,and sequence alignments were conducted for intron length polymorphisms exploitation. Multiplex PCR was established for the identification of P. ginseng and P. quinquefolius with their specific primers,which were designed respectively based on their insertion sequences. And the limit of detection of the multiplex PCR was also determined. Result: The insertion/deletion sequences were exploited in mitochondrial cox Ⅱ. Under the established multiplex PCR assay,P. ginseng generated a 729 bp specific band,while P. quinquefolius yielded a 141 bp specific amplicon,and the mixture of the two species yielded both729 bp and 141 bp fragments. The established multiplex PCR assay could detect 0. 1% of intentional adulteration of P. quinquefolius into P. ginseng,with down to 0. 001 ng of genomic DNA. Conclusion: The established multiplex PCR assay can accurately identify P. ginseng and P. quinquefolius from different sources,without the optimization of reaction system and the introduction of additional mismatches,so as to provide a new molecular marker method for identifying botanical origin of P. ginseng and P. quinquefolius.