基于DNA条形码和高分辨率熔解曲线技术快速鉴别肉苁蓉  

Rapid identification of Cistanches Herba based on DNA barcoding and high resolution melting

作　　者：过立农[1] 刘杰[1,2] 赵春艳[3] 昝珂 郑健[1] 马双成[1] 陈艳菲[4] 李昀铮 GUO Li-nong;LIU Jie;ZHAO Chun-yan;ZAN Ke;ZHENG Jian;MA Shuang-cheng;CHEN Yan-fei;LI Yun-zheng(National Institutes for Food and Drug Control，Minzu University of China，Sichuan Institute for Food and Drug Control，Taiyuan Institute for Food and Drug Control，Songyuan Institute for Food and Drug Contro)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]中央民族大学,北京100081 [3]四川省食品药品检验检测院,成都611731 [4]太原市食品药品检验所,太原030000 [5]松原市食品药品检验所,松原138000

出　　处：《药物分析杂志》2018年第4期665-671,共7页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:基于DNA条形码技术建立肉苁蓉样品的高分辨率熔解曲线(HRM)鉴定方法。方法:通过聚合酶链式反应(PCR)对肉苁蓉的ITS和ITS2序列进行扩增;利用MEGA 6.06软件通过建树法进行NJ树聚类分析;通过ITS2序列利用Primer Premier 5软件进行引物设计;对经设计、合成的8对引物通过HRM实验进行筛选;建立肉苁蓉的HRM鉴定方法。结果:通过ITS和ITS2序列的NJ树聚类分析可以有效区分3种肉苁蓉(肉苁蓉、管花肉苁蓉及采自乌兹别克斯坦的肉苁蓉相近种),且肉苁蓉和管花肉苁蓉各自的栽培品与野生品不存在明显差异;经过Primer Premier 5软件进行引物设计共选出8对引物进行合成;通过HRM预实验筛选出引物Ysr-2为最佳实验引物;确立了有效鉴定3种肉苁蓉样品的HRM方法。结论:通过DNA条形码和HRM技术均能有效鉴定3种肉苁蓉样品,且结果一致,起到互相验证的作用,为肉苁蓉及其他中药民族药品种的快速鉴定提供了可以参考的依据。Objective：To establish an identification method of high resolution melting（HRM）for Cistanches Herba based on DNA barcoding.Method：ITS and ITS2 sequences of Cistanches Herba were amplified with polymerase chain reaction（PCR）and the spectral of clustering analysis tree was drawn through the method of Neighbour Joining with the software of MEGA 6.06.Then primers were designed using the software of Primer Premier 5 based on ITS2 sequence,the best of which was screened from 8 through the HRM experiment.Finally,the identification method of HRM for Cistanches Hebra was established.Results：Three kinds of Cistanche（Cistanche tubulosa,Cistanche deserticola and Cistanche imported from Uzbekistan）could be effectively distinguished by Neighbour Joining clustering analysis of ITS and ITS2 sequences,and there were no differences between the wild and cultivated samples of Cistanche deserticola and Cistanche tubulosa.The HRM experiment confirmed Ysr-2 as the best primer and the HRM identification method was established.Conclusion：The methods of DNA Barcoding and HRM both could effectively identify three kinds of Cistanche and the results were consistent and verified each other.The rapid identification technology of Cistanches Herba could also provide the reference basis for other traditional medicines.

关 键 词：肉苁蓉 聚合酶链式反应(PCR) 内部转录间隔区2(ITS2) 内部转录间隔区(ITS) DNA条形码 高分辨率熔解曲线(HRM) 

分 类 号：R917[医药卫生—药物分析学]