基于特异性PCR的梅花鹿茸和马鹿茸区分

Differentiation of Cervi Cornu Pantotrichum Derived from Cervus nippon and Cervus elaphus by Specific PCR

目的:开发及验证梅花鹿和马鹿茸的特异性聚合酶链式反应(PCR)筛选方法。方法:采集梅花鹿茸、马鹿茸、鹿茸混淆品、香港市场流通的鹿类产品、非鹿科动物、植物和真菌类样品。对鹿类线粒体基因组进行排序,设计及筛选特异性引物,采用特异性PCR对样品进行区分,并进行条件优化和方法学验证。结果:共收集77份样品,对87份线粒体基因组DNA排序并设计9个特异性引物组合。经验证后,CNIP-f/CNIP-r和CELA-f/CELA-r分别选作梅花鹿和马鹿的特异性引物组合,通过PCR可获得223 bp的梅花鹿和248 bp的马鹿特异性条带,作为区分梅花鹿茸和马鹿茸的依据。结论:该方法能作为中药鉴别的补充方案,用于质量控制中涉及动物制品的种属,进一步完善鹿茸饮片鉴别体系。

Objective:To develop and verify a specific polymerase chain reaction(PCR)method for differentiation of Cervi Cornu Pantotrichum derived from Cervus nippon Temminck and C.elaphus Linnaeus.Methods:Cervi Cornu Pantotrichum derived from C.nippon and C.elaphus,the adulterants,deer-derived products in Hong Kong market,non-Cervidae animal samples,plant samples,and fungal samples were collected.According to the Cervidae mitochondrial genome DNA sequences,specific primers were designed and screened.The testing conditions were optimized and the method was validated.Results:A total of 77 samples were collected.Alignment of 87 Cervidae mitochondrial genomes was performed and 9 specific primer combinations were designed.After method validation,CNIP-f/CNIP-r and CELA-f/CELA-r were selected as the specific primer combinations for Cervi Cornu Pantotrichum derived from C.nippon and C.elaphus,respectively.A 223 bp C.nippon sample-specific band and a 248 bp C.elaphus sample-specific band were yielded by PCR,which were the bases for distinguishing the Cervi Cornu Pantotrichum samples derived from the two species.Conclusion:The method boosts the identification of Cervi Cornu Pantotrichum Decoction Pieces and can be used for the identification of animal medicinals derived from different species.