多重位点特异性PCR同时鉴别酸枣仁及其掺伪品  

Simultaneous Identification of Ziziphi Spinosae Semen and Its Adulterants by Multiplex Allele-specific PCR

作　　者：李柯帆 索晓雄 李晓兰 杜晨晖 闫艳[2] 裴香萍 LI Kefan;SUO Xiaoxiong;LI Xiaolan;DU Chenhui;YAN Yan;PEI Xiangping(College of Traditional Chinese Medicine(TCM)and Food Engineering,Shanxi University of Chinese Medicine,Jinzhong 030619,China;Modern Research Center for TCM,Shanxi University,Taiyuan 030006,China)

机构地区：[1]山西中医药大学中药与食品工程学院,山西晋中030619 [2]山西大学中医药现代研究中心,太原030006

出　　处：《中国实验方剂学杂志》2023年第2期141-148,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：山西省中药现代化振东专项基金项目(2016ZD0104);国家自然科学基金青年科学基金项目(81603251)。

摘　　要：目的:优化并建立多重位点聚合酶链式反应(PCR)体系同时鉴别酸枣仁、枳椇子和理枣仁及不同掺伪量,解决酸枣仁药材商品及其制剂的掺伪难题。方法:通过分析比对酸枣仁及其伪品的内部转录间隔区(ITS)序列差异找到特异性单核苷酸多态性(SNP)位点,设计特异性鉴别引物,通过优化退火温度、循环次数及引物浓度,评估不同聚合酶种类和不同PCR仪等扩增条件对不同来源的酸枣仁、枳椇子及理枣仁样品进行特异性扩增,根据特异性扩增条带大小进行鉴别,并对最低检测限和掺伪检出限进行研究。结果:在退火温度为63℃,循环次数为23次时,酸枣仁、枳椇子和理枣仁分别扩增出549、169、389 bp的特异性条带。该方法对酸枣仁和理枣仁的最低检测限为0.24 ng,枳椇子为1.2 ng,对酸枣仁、枳椇子和理枣仁的掺伪检出限分别为0.5%、2%和2%。结论:该文建立的多重位点特异性PCR鉴别方法能同时准确鉴别酸枣仁、枳椇子和理枣仁,对解决酸枣仁药材掺伪问题提供基础研究,为控制酸枣仁药材质量安全及临床应用提供参考。Objective:To optimize and establish a multiplex polymerase chain reaction(PCR)system to simultaneously identify Ziziphi Spinosae Semen(ZSS),Hovenia acerba semen(HAS),and Ziziphi Mauritianae Semen(ZMS),etermine their content to solve the problem of adulteration of ZSS pieces and its preparations.Method:After the analysis and comparison of the internal transcribed spacer(ITS)sequence differences of ZSS and its adulterants,specific single nucleotide polymorphism(SNP)sites were found,and specific primers for identification were designed.The samples of ZSS,HAS,and ZMS from different sources were specifically amplified under the conditions of optimized annealing temperature,the number of cycles,and concentration of primers,as well as different polymerases and PCR systems after evaluation.Identification was carried out according to the size of specific amplification bands,and the lower limit of detection(LOD)and adulteration LOD were studied.Result:When the annealing temperature was 63℃ and the number of cycles was 23,549,169,389 bp specific bands were amplified from ZSS,HAS,and ZMS.The lower LOD of this method was 0.24 ng and 1.2 ng for ZSS and HAS,respectively.The adulteration LOD for ZSS,HAS,and ZMS was 0.5%,2%,and 2% respectively.Conclusion:The established multiplex allele-specific PCR identification method can accurately identify ZSS,HAS,and ZMS at the same time,which can provide a basis for solving the problem of adulteration of ZSS and references for controlling the quality,security,and clinical application of ZSS.

关 键 词：酸枣仁 枳椇子 理枣仁 多重位点特异性聚合酶链式反应(PCR) 鉴别 掺伪 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学] R22R2-031R33