目的:建立基于高通量测序技术的11 种乌头属药材混合粉末分子鉴定方法。方法:采用高通量测序技术,对11 种乌头属药材混合粉末样品中的总DNA 经提取,对ITS2 片段进行PCR 扩增,利用IlluminaMiSeq 平台对DNA 片段进行双端测序,最后采用FLASH、QIIME、GraPhlAn 及MEGA 6.0 软件对序列进行整理并聚类分析,鉴定混合粉末中的物种。结果:混合样品中得到高质量的ITS2 序列共82 348 条, 样品中乌头属物种含有序列69 814 条,比对出其中的8 个物种,即样品中含有草乌、川乌、阴山乌头、华北乌头、瓜叶乌头、毛叶乌头、花葶乌头、高乌头;未能比对出黄花乌头、松潘乌头、川鄂乌头3 种乌头属药材。结论:以ITS2 作为条形码,采用高通量测序技术,可以鉴定出乌头属药材混合粉末中的8 个物种,为混合药材的鉴定提供新思路。
Objective: To establish an high-throughput sequencing method for molecular identification of mixed powders of 11 species of Aconitum medicinal materials. Method: Using high-throughput sequencing technology, total DNA from 11 kinds of Aconitum medicinal powder samples was extracted following PCR amplification of ITS2 fragments. Paired-end sequencing of the DNA fragment was performed using IlluminaMiSeq platform. Finally,using FLASH,QIIME,GraPhlAn and MEGA 6.0 software,the sequence was sorted out and the species in the mixed powder were identified by cluster analysis. Results: A total of 82 348 high-quality ITS2 sequences were obtained in the mixed samples. The Aconitum species contained 69 814 sequences. 8 species were found including Radix Aconiti kusnezoffii,Radix Aconiti Carmichaelii ,Radix Aconiti Yinschanicum,Radix Aconiti jeholense,Radix Aconiti hemsleyanum,Radix Aconiti carmichaelii,Radix Aconiti scaposum and Radix Aconiti sinomontanum. But Radix Aconiti coreanum ,Radix Aconiti sungpanense and Radix Aconiti henryi were not identified successfully. Conclusion: Using ITS2 as a barcode,the high-throughput sequencing technology can identify 8 species in the mixed powder of Aconitum medicinal materials,providing new ideas for the identification of mixed medicinal materials.
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