基于DNA条形码和HRM技术鉴别肉苁蓉疑似伪品

Identification of suspected fake Cistancehe Hebra based on DNA barcoding and high resolution melting

目的:基于DNA条形码技术及高分辨率熔解曲线(HRM)方法鉴定肉苁蓉药材的基原。方法:通过聚合酶链式反应(PCR)对肉苁蓉样品的ITS2序列进行扩增;将待鉴定肉苁蓉样品的ITS2序列与Genbank数据库进行BLAST比对;利用MEGA 6.06软件通过建树法进行NJ树聚类分析;通过HRM方法快速鉴定肉苁蓉药材基原。结果:21份待鉴定肉苁蓉样品ITS2序列的BLAST比对结果中,11份样品与沙苁蓉高度一致,其余10份样品与《中华人民共和国药典》2015年版一部收载品种肉苁蓉(Cistanche deserticola)或管花肉苁蓉(Cistanchetubulosa)高度一致;通过ITS2序列的NJ聚类分析,将21份待鉴定肉苁蓉样品分别聚类到沙苁蓉、管花肉苁蓉、盐生肉苁蓉、肉苁蓉4个种;通过HRM方法鉴定肉苁蓉药材样品,鉴定结果与NJ聚类分析结果一致。结论:通过DNA条形码和HRM技术均能有效鉴定不同种的肉苁蓉药材样品,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参考依据。

Objective:To identify origin of Cistancehe Hebra based on DNA barcoding technology and high resolution melting(HRM). Method:ITS2 sequences of Cistancehe Hebra were amplified with polymerase chain reaction(PCR)and BLAST with Genbank. Phylogenetic analysis with neighbour joining tree was performed using the software of MEGA 6.06. The rapid identification of Cistancehe Hebra was carried out based on the method of HRM. Results:BLAST results of ITS2 sequences from the 21 samples under identification indicated that 11 samples were highly consistent with Cistanche sinensis and 10 samples highlyy consistent with Cistanche deserticola and Cistanche tubulosa. NJ phylogenetic analysis classified the 21 samples into 4 categories including Cistanche sinensis,Cistanche tubulosa,Cistanche salsa and Cistanche deserticola. Results of HRM identification were consistent with the NJ phylogenetic analysis. Consult:DNA barcoding and HRM both can effectively identify the Cistancehe Hebra,the results were consistent and mutual supporting,which provides the reference basis for rapid identification of other traditional medicines.