基于COⅠ序列的阿胶原材料及其混伪品的DNA条形码鉴定研究

目的：采用细胞色素C氧化酶亚基1（cytochrome C oxidase subunit 1，COⅠ）基因片段序列鉴定的方法鉴别中药阿胶的原料驴皮及其混伪品马皮、骡皮、牛皮。方法：优化DNA提取试剂盒的通用方法进行总DNA的提取，针对马科动物驴的基因序列变异位点优化COⅠ通用引物及PCR扩增条件，PCR扩增产物双向测序，测得的序列与GenBank下载的基因序列Blast比对分析样品的基原；采用Mega 6.0软件计算序列的碱基组成、种内和种间K2P（Kimura-2-parameter）的遗传距离，采用邻接法（neighbor-joining method）构建系统树，进一步对驴皮及其混伪品进行基原鉴别。结果：22份来自马科和牛科动物的驴、马、牛的皮样品的基因序列与GenBank序列Blast比对结果一致性均大于99%。采用邻接法构建的系统树结果显示驴、马、牛的皮样品具有较好的单系性，均分别独立聚为1个分支，可以显著区分；牛科和马科动物样品的K2P种间平均遗传距离均远大于种内平均遗传距离。结论：利用优化后的COⅠ片段扩增引物及PCR反应程序可作为鉴别阿胶原料DNA条形码物种鉴定的有效方法，为阿胶原料的真伪鉴别提供了科学的鉴定方法。

Objective:To identify donkey hide, the raw material of Asini Colla Corii, and its counterfeits including hide of horse, mule and bovine, based on cytochrome C oxidase subunit 1(COⅠ) barcoding.Methods:The genomic DNA fragments were extracted by optimized methods through universal COⅠ primer and polymerase chain reaction(PCR) condition.The PCR products were bi-directionally sequenced.Sequences were blasted with the sequences from the GenBank.The basic group composition of COⅠ sequence, the inter-and intra-specific variations of COⅠ sequences were analyzed and compared based on Kimura-2-parameter(K2P) distances, and the neighbor-joining method was used to reconstruct phylogenetic tree by Mega 6.0 software to identify donkey hide and its counterfeits.Results:The consistencies of 22 samples were all above 99% by Blast comparison with the data from GenBank.The phylogenetic tree reconstructed by neighbor-joining method could distinguish donkey hide from hide of horse, mule and bovine because of the K2P distance of intra-distance was much larger than that of inter-distance.Conclusion:COⅠ DNA barcoding coupled with PCR offered an efficienct method for origin identification of the raw material of Asini Colla Corii.

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