School of Pharmacy, Guizhou University of Traditional Chinese Medicine; School of Pharmacy/School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine.
School of Pharmacy/School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine.
J Vis Exp. 2024 Nov 1(213). doi: 10.3791/66925.
Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.
药用植物是全球有价值的资源,在世界范围内被用于保持健康和治疗疾病;然而,掺假的存在阻碍了它们的发展。DNA 条形码是一种通过标准 DNA 区域进行物种鉴定的技术,有助于快速准确地鉴定传统药用植物。DNA 条形码的过程包括六个基本步骤:1)处理药用植物,2)使用离心柱法从药用植物中提取高质量的总 DNA,3)用植物通用引物扩增目标 DNA 区域内转录间隔区 2(ITS2)并进行 Sanger 测序,4)拼接和对齐序列以获得目标序列,5)将条码序列与条码库进行比对以进行鉴定,6)对齐序列,比较种内和种间变异,构建系统发育邻接法树。结果表明,通用引物可以扩增目标区域。基本局部比对搜索工具(BLAST)显示鉴定的百分比为 100%,邻接法树表明拼接序列与 A. sinensis OR879715.1 分支聚类,分支支持值为 100。本方案为应用 DNA 条形码技术作为鉴定药用植物和掺杂物的有效方法提供了参考。