Ye Zi, Lu Ye, Wang Zheng-Tao, Xu Hong, Hu Zhi-Bi
Zhongguo Zhong Yao Za Zhi. 2014 Oct;39(20):3928-35.
Isolation of high-quality genomic DNA from dried and processed crude drug is the key for the DNA identification of Dendrobii Caulis. The DNA extract of Dendrobii Caulis was firstly compared using different method to isolate genomic DNA from dried and processed crude drug, including commercial DNA extracted kit and CTAB method. Using modified CTAB method (extracted from large samples), the genomic DNA was successfully isolated from Dendrobii Caulis, including Huangcao and Fengdou. The ITS regions were amplified using the purified DNA as template, and then cloned and sequenced. These ITS sequences were compared with data from Genbank database and our lab, 14 Dendrobium species and five similar species were identified from "Huangcao" and "Huangcao" slice, while six species and three similar species from "Fengdou" according to their sequence similarity. The study demonstrated that the dried Dendrobii Caulis could be identified using DNA molecular method, which could overcome deficiencies and limitations of traditional identification method and has a certain application prospects.
从干燥及加工后的中药材中分离高质量的基因组DNA是石斛茎DNA鉴定的关键。首先使用不同方法从干燥及加工后的中药材中分离基因组DNA,对石斛茎的DNA提取物进行比较,包括商业DNA提取试剂盒和CTAB法。采用改良的CTAB法(从大量样品中提取),成功从石斛茎(包括黄草和枫斗)中分离出基因组DNA。以纯化后的DNA为模板扩增ITS区域,然后进行克隆和测序。将这些ITS序列与Genbank数据库和本实验室的数据进行比较,根据序列相似性从“黄草”和“黄草片”中鉴定出14种石斛属植物和5种近缘物种,从“枫斗”中鉴定出6种石斛属植物和3种近缘物种。研究表明,采用DNA分子方法可对干燥的石斛茎进行鉴定,该方法能够克服传统鉴定方法的不足和局限性,具有一定的应用前景。