乌梢蛇配方颗粒与常见三种伪品的多重PCR鉴别研究  

Multiplex PCR identification of Zaocys dhumnades formula granules and three common counterfeit products

作　　者：宋叶 罗宇琴 李国卫 冼乐尧 谭斯尹 范耀耀 罗怡靖 陈向东 孙冬梅 SONG Ye;LUO Yuqin;LI Guowei;XIAN Leyao;TAN Siyin;FAN Yaoyao;LUO Yijing;CHEN Xiangdong;SUN Dongmei(Guangdong Yifang Pharmaceutical Co.,Ltd.,Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Formula Granule,Foshan 528244,China;Guangzhou University of Chinese Medicine,Guangzhou 510000,China)

机构地区：[1]广东一方制药有限公司,广东省中药配方颗粒企业重点实验室,广东佛山528244 [2]广州中医药大学,广州510000

出　　处：《中国药品标准》2024年第4期321-329,共9页Drug Standards of China

基　　金：广东省基础与应用基础研究基金项目(2021A1515110790);2022年佛山市南海区重点领域科技攻关专项(南科﹝2023﹞20号-18)。

摘　　要：目的:建立乌梢蛇、王锦蛇、百花锦蛇、灰鼠蛇药材及水提物四重位点特异性聚合酶链反应(PCR)鉴别方法,为配方颗粒的掺伪鉴别提供依据。方法:以线粒体细胞色素C氧化酶1号(CO1)基因为靶基因,设计特异性引物,优化引物最适退火温度、循环次数、聚合酶种类,并用该方法进行混合样品的检测。结果:乌梢蛇、王锦蛇、百花锦蛇、灰鼠蛇聚合酶为Multiplex PCR 5×Master Mix,退火温度为60℃,循环次数为35次时分别扩增出133、180、247、197 bp的特异性条带,空白对照无条带。该方法能同时、准确地鉴定出混合样品的蛇源成分。结论:本方法可同时、准确、快速地鉴定乌梢蛇、王锦蛇、百花锦蛇、灰鼠蛇样品,并适合标准汤剂和配方颗粒样品鉴别。Objective:To establish a polymerase chain reaction(PCR)method to accurately discriminate the crude materials and aqueous extract of Zaocys dhumnades,Elaphe carinata,Elaphe meollendorff and Ptyas korros.Methods:Specific primers were designed using mitochondrial Cytb gene(CO1)as a target gene,and annealing temperature,number of cycles and the type of DNA polymerases were optimized.The mixed samples were detected by this method.Results:By this multiplex allele-specific PCR identification method,135,182,246 and 197 bp of specific fragments were amplified from DNA templates of Zaocys dhumnades,Elaphe carinata,Elaphe meollendorffi and Ptyas korros,respectively,following the conditions:cycle number of 35,annealing temperature of 62℃.The adulterants and the blank control showed no bands.The method could simultaneously and accurately identify the snake-derived components in the mixed samples.Conclusion:The method can be used to identify the samples of Zaocys dhumnades,Elaphe carinata,Elaphe meollendorffi and Ptyas korros simultaneously,accurately and rapidly,and is suitable for the identification of standard decoctiond and formula granules samples.

关 键 词：乌梢蛇 王锦蛇 百花锦蛇 灰鼠蛇 配方颗粒 多重位点特异性聚合酶链反应法 

分 类 号：R921.2[医药卫生—药学]