多重连接探针扩增技术鉴别酸枣仁及混伪品的研究  

Identification of Ziziphus jujuba var. spinosa Seeds and its Adulterants by Multiplex Ligation-dependent Probe Amplification Technique

作　　者：王文斌 莫静 程华春 胡志刚[2] 杜巍[4] 汪波 WANG Wen-bin;MO Jing;CHENG Hua-chun;HU Zhi-gang;DU Wei;WANG Bo(Hubei Institute for Drug Control,Wuhan 430075,China;Hubei University of Chinese Medicine,Wuhan 430065,China;School of Life Sciences,Hubei University,Wuhan 430062,China;College of Life Sciences,Wuhan University,Wuhan 430072,China)

机构地区：[1]湖北省药品监督检验研究院,湖北武汉430075 [2]湖北中医药大学,湖北武汉430065 [3]湖北大学生命科学学院,湖北武汉430062 [4]武汉大学生命科学学院,湖北武汉430072

出　　处：《中药材》2022年第4期818-823,共6页Journal of Chinese Medicinal Materials

基　　金：国家自然科学基金项目(81973429);湖北省科技重大专项(2020ACA007);湖北省自然科学基金重点类项目(2020CFA024)

摘　　要：目的:针对市场上出现的酸枣仁药材掺假现象,开发一种快速鉴别酸枣仁药材是否含有掺伪品的方法。方法:基于多重连接探针扩增技术(MLPA),针对酸枣仁与理枣仁ITS2序列205位点处A/G变异,以及枳椇子170位点分别设计酸枣仁、理枣仁与枳椇子的MLPA探针,MLPA扩增后依据熔解曲线Tm值差异来进行鉴别,并考察了特异性、敏感度、掺伪比等内容,以验证该方法的可行性。结果:酸枣仁、理枣仁和枳椇子分别与特异性探针在85.56℃、87.26℃和83.93℃处出现单一扩增峰,表明探针特异性好;通过不断稀释DNA模板,仍旧可以检测出0.05 ng酸枣仁DNA量,同样酸枣仁中掺入10%的伪品均可被有效检出,表明该方法具有高灵敏度。结论:该研究建立的酸枣仁药材及其混伪品鉴别方法具有特异性强、灵敏度高的特点,对于解决酸枣仁药材掺伪问题具有良好的推广应用价值。Objective:To develop a rapid identification method for adulteration of Ziziphus jujuba var.spinosa seeds in the market.Methods:Based on the multiplex ligation-dependent probe amplification technique(MLPA),the MLPA probes were designed according to the A/G variation of 205 th site in the ITS2 sequence of the seeds of Ziziphus jujuba var.spinosa and Ziziphus mauritiana and the 170 th site of Hovenia acerba seeds.After MLPA amplification,the differences of melting curve Tm value were used for identification,the specificity,sensitivity and adulteration ratio were investigated to verify the feasibility of this method.Results:The seeds of Ziziphus jujuba var.spinosa,Ziziphus mauritiana,Hovenia acerba showed a single amplification peak at 85.56,87.26,83.93℃,respectively,indicating that the specificity of the probe was good.By continuously diluting the DNA template,the amount of DNA in 0.05 ng Ziziphus jujuba var.spinosa seeds could still be detected,and 10%of the adulterants in Ziziphus jujuba var.spinosa seeds could be effectively detected,indicating that this method had high sensitivity.Conclusion:In this study,the identification method of Ziziphus jujuba var.spinosa seeds and its adulterants is established,which has the characteristics of strong specificity and high sensitivity.It has good popularization and application value to solve the adulteration problem of Ziziphus jujuba var.spinosa seeds.

关 键 词：酸枣仁 理枣仁 枳椇子 多重连接探针扩增技术 鉴别 

分 类 号：R282.5[医药卫生—中药学]