基于ITS2条形码的市售延胡索鉴别

Identification of Commercially Available Corydalis yanhusuo by ITS2 Barcode

目的:利用DNA条形码技术对市售的经过蒸煮等加工的延胡索饮片进行分子鉴定,以评价内转录间隔区2(internal transcribed spacer 2,ITS2)序列对延胡索饮片的鉴别能力。方法:采用改良后的试剂盒法提取样品DNA,用ITS2序列引物进行PCR扩增后测序,采用CodonCode Aligner软件对测序峰图进行拼接、校对,采用MEGA 7.0软件进行种内、种间变异比对及Kimura 2-parameter(K2P)遗传距离分析,并基于邻接法构建系统聚类树。结果:约80%的样品的ITS2序列被成功扩增与测序,系统聚类树显示,所得延胡索ITS2序列和参考序列紧密聚合,具有良好的单系性,能够明显区分混伪品。结论:ITS2条形码可以准确鉴别延胡索不同加工品及其常见混伪品,为延胡索的鉴定提供分子生物学依据。

Objective:To evaluate the identification capacity of the internal transcribed spacer2(ITS2)sequence on the commercially available Corydalis yanhusuo decoction pieces through molecular identification of processed C.yanhusuo decoction pieces by DNA barcoding technique.Methods:DNA samples were extracted by the modified kit assay.After PCR amplification with ITS2 sequence primers,sequencing was performed,and obtained sequences were assembled using CodonCode Aligner.The genetic distances and intraspecific or interspecific variation were computed by MEGA 7.0 in accordance with the Kimura 2-parameter(K2P)model and the phylogenetic tree was established by Neighbor-joining(NJ)method.Results:About80%of the ITS2 regions were PCR-amplified and sequenced successfully.The NJ tree showed that the ITS2 regions obtained were closely aggregated with the reference regions,showing good unilineality,which can be used to distinguish between C.yanhusuo and its adulterants.Conclusion:ITS2 barcode can be used for the identification of the processed products of C.yanhusuo and their adulterants correctly,which provides a molecular biological basis for identification of the processed products of C.yanhusuo.