基于DNA条形码和高分辨率熔解曲线技术快速鉴别肉苁蓉

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.