基于巢式PCR方法联合Sanger测序技术对大黄庶虫丸中水蛭成分真伪的鉴定

目的 基于DNA条形码技术建立一种可以实现对大黄庶虫丸中水蛭成分真伪品鉴定的方法。方法 首先以COⅠ通用引物对大黄庶虫丸中成药DNA进行一轮聚合酶链式反应(PCR)扩增,随后采用自行设计和筛选出的水蛭通用引物对一轮PCR反应液进行二轮PCR扩增,联合Sanger测序技术,对二轮PCR扩增产物测序,最后对测序结果峰图采用seqman软件进行序列拼接,将拼接后的DNA序列在NCBI数据库中用BLAST在线比对软件进行比对判定,进而鉴定水蛭成分真伪。结果 本实验建立的巢氏PCR方法联合Sanger测序技术通过了对9份经COⅠ通用引物鉴定的水蛭干药材的验证,结果验证两方法鉴定结果一致。同时采用本研究建立的巢氏PCR方法联合Sanger测序技术对收集的14批大黄庶虫丸进行鉴定,共有5批鉴定出含有伪品水蛭成分。结论 本实验建立的的巢氏PCR方法联合Sanger测序技术能够实现对大黄庶虫丸中水蛭成分真伪品的鉴定,对中成药的质量控制具有一定的意义。

Authenticity Identification of Leech Components in Dahuang Zhechong Pills Based on Nested PCR Methods Combined with Sanger Sequencing Technology

OBJECTIVE To establish a method based on DNA barcode technology to identify the authenticity of leeches in Chinese patent medicine Dahuang Zhechong Pills. METHODS First, a round of PCR amplification by universal primers targeting the COⅠ gene was performed on the DNA of Dahuang Zhechong Pills. Subsequently, the products of the first round of PCR amplification were used as templates to perform the second round of PCR with self-designed universal primers for leech. The final PCR reaction solution was sequenced and the sequencing results were then sequence stitched using seqman software. The spliced DNA sequence was determined by BLAST alignment in the NCBI database, and then the authenticity of the leech was identified. RESULTS The nested PCR method established in this study was used to identify nine dried leeches, and the identification results were consistent with the identification results of COⅠ universal primers. At the same time, the nested PCR method was also used to identify 14 batches of Dahuang Zhechong pills which were made by different manufacturers, and five of them were found to contain fake leeches. CONCLUSION The nested PCR method established in this paper combined with Sanger sequencing technology can realize the identification of the authenticity of the leeches in the Chinese patent medicine Dahuang Zhechong Pills, which has certain significance for the quality control of Chinese patent medicines.