柴胡属药用植物多重实时荧光PCR检测方法的建立  

Establishment of multiplex real-time fluorescence PCR detection method for Bupleurum

作　　者：张全芳[1] 范阳阳[1] 刘艳艳[1] 陈雪燕[1] 谭晴晴[1] 胡悦 刘国霞[1] 汪冰 林永强 步迅[1] ZHANG Quanfang;FAN Yangyang;LIU Yanyan;CHEN Xueyan;TAN Qingqing;HU Yue;LIU Guoxia;WANG Bing;LIN Yongqiang;BU Xun(Biotechnology Research Center,Applied Life Key Sciences Lab,Shandong Academy of Agricultural Sciences,Jinan 250100,China;Shandong Institute for Food and Drug Control,Key Laboratory for Quality Evaluation of Rubber Products of the State Drug Administration,Jinan 250101,China)

机构地区：[1]山东省农业科学院生物技术研究中心,山东省农业科学院应用生命科学实验室,山东济南250100 [2]山东省食品药品检验研究院,国家药品监督管理局胶类产品质量评价重点实验室,山东济南250101

出　　处：《药学研究》2019年第12期693-696,708,共5页Journal of Pharmaceutical Research

基　　金：山东省重点研发计划(No.2018GSF119002、GG201809150023);山东省创新公共服务平台计划(No.2018JGX111);山东省农业科学院农业科技创新工程(No.CXGC2018D03)

摘　　要：目的用实时荧光定量聚合酶链式反应(qPCR)方法测定正品柴胡,为柴胡的分子鉴定提供依据。方法从柴胡根茎中提取总DNA,根据ITS2序列,设计合成针对南柴胡和北柴胡的特异性引物和探针以及柴胡的通用引物与探针,通过对荧光聚合酶链式反应反应体系和反应条件的优化筛选,建立了多重实时荧光聚合酶链式反应方法,在同一聚合酶链式反应反应体系中可以同时鉴别南、北柴胡。结果结果表明本试验设计的引物和探针具有很好的物种特异性,且灵敏度高,DNA检出限为0.08 ng。对46份样品检测,其中17份检出北柴胡源性成分,5份为南柴胡,其他样品均检测为伪品柴胡,检测结果与DNA测序结果一致。结论该方法可以有效地鉴别出南、北柴胡源性成分,及伪品柴胡,对于保证柴胡药材的质量及用药安全具有重要意义。Objective To determine authentic Bupleurum by qPCR method,and provide basis for molecular identification of Bupleurum.Methods The total DNA was extracted from the root of Bupleurum species.Based on the ITS2 sequence,specific primers and probes targeting Bupleurum chinense DC.and Bupleurum scorzonerifolium Willd.,and universal primers and probes of Bupleurum species were designed.The conditions were optimized and screened.A multiplex real-time fluorescent PCR method was established,which could identify Bupleurum chinense DC.and Bupleurum scorzonerifolium Willd.simultaneously in the same PCR reaction system.Results The results showed that the primers and probes designed in this experiment have good species specificity and high sensitivity,and the DNA detection limit is 0.08 ng.Forty-six samples were tested,17 of them were derived from Bupleurum chinense DC.,5 were from Bupleurum scorzonerifolium Willd.,and other samples were detected as fake Bupleurum.The test results were consistent with DNA sequencing results.Conclusion This method can effectively identify the source components of Bupleurum chinense DC.and Bupleurum scorzonerifolium Willd.and fake Bupleurum.It is of great significance to ensure the quality and safety of Bupleurum.

关 键 词：ITS2基因 多重实时荧光聚合酶链式反应 南柴胡 北柴胡 

分 类 号：R282.5[医药卫生—中药学]