基于DNA条形码和HRM技术建立紫草药材的RFLP-HRM鉴别方法

RFLP-HRM identification method of Arnebiae Radix based on DNA barcoding and high resolution melting

目的:基于DNA条形码技术和高分辨率熔解曲线(HRM)技术鉴别紫草市场样品的真伪并建立紫草药材的RFLP-HRM鉴别方法。方法:通过聚合酶链式反应(PCR)对紫草药材的ITS2序列进行扩增;利用MEGA 6.06软件对紫草药材的ITS2序列进行NJ树聚类分析和遗传距离分析;对市场中紫草非药典品进行基原确证;针对紫草药材的ITS2序列利用Primer Premier 5软件进行引物设计;对经设计、合成的引物通过HRM实验进行筛选;建立紫草药材的RFLP-HRM鉴定方法。结果:通过对紫草药材的ITS2序列进行NJ树聚类分析和遗传距离分析,可以有效区分新疆紫草、疏花软紫草、黄花软紫草及市场上非药典来源的紫草药材(紫草非药典品);经过Primer Premier 5软件进行引物设计共选出4对引物进行合成;通过HRM实验筛选出引物Zcf-1为最佳实验引物;确立了有效鉴别紫草市场药材的RFLP-HRM方法。结论:通过DNA条形码和HRM技术均能有效鉴别紫草市场药材真伪,且结果一致,起到互相验证的作用,建立了紫草市场药材的RFLP-HRM鉴定方法,为紫草及其他中药民族药品种的快速鉴定技术提供了可以参考的依据。

Objective:To establish an identification method of restriction fragment length polymorphism-high resolution melting(RFLP-HRM)for Arnebiae Radix from medicine market based on DNA barcoding and high resolution melting(HRM).Methods:Internal transcribed spacerⅡof nuclear ribosomal DNA(ITS2)sequences of Arnebiae Radix were amplified with polymerase chain reaction(PCR).MEGA 6.06 software was used to perform NJ tree cluster analysis and genetic distance analysis on the ITS2 sequence of Arnebiae Radix.Originalplant confirmation of non-pharmacopoeial Arnebiae Radix from the market was performed.The primers were disigned using the software of Primer Premier 5 based on ITS2 sequence.The best one from 4 designed primers was screened through the HRM experiment.The identification method of RFLP-HRM for Arnebiae Radix was established.Results:The neighbour joining clustering analysis and genetic distance analysis of ITS2 sequences could effectively distinguish among the different species for this study.The HRM experiment confirmed Zcf-1 was the best primer.The RFLP-HRM method to effectively identify Arnebiae Radix from medicine market was established.Conclusion:The methods of DNA Barcoding and HRM both can effectively identify the Arnebiae Radix in this study,and the results are consistent and mutual authentication.The RFLP-HRM rapid identification method of Arnebiae Radix from medicine market has been established,which provides the reference basis for other traditional medicines.