Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan 250062, China; School of Medicine and Life Sciences, University of Jinan, Shandong Academy of Medical Sciences, Jinan 250062, China.
Shandong Key Laboratory of Energy Genetics, CAS Key Laboratory of Biofuels and Bioinformatics Group of Single Cell Center, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China.
Genomics Proteomics Bioinformatics. 2014 Jun;12(3):137-43. doi: 10.1016/j.gpb.2014.03.002. Epub 2014 May 14.
Traditional Chinese medicine (TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients, which often include some mis-identified herbal materials, adulterants or even some biological contaminants. For biological ingredient analysis, the efficiency of DNA extraction is an important factor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation, Liuwei Dihuang Wan (LDW), as an example to develop a TCM-specific DNA extraction method. An optimized cetyl trimethyl ammonium bromide (CTAB) method (TCM-CTAB) and three commonly-used extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer (ITS2) and the chloroplast genome trnL intron was carried out. The results have shown that PCR amplification was successful only with template of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3-4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting TCM-CTAB method could facilitate follow-up DNA-based examination of TCM preparations.
中药制剂广泛用于医疗保健和临床实践。到目前为止，中药制剂质量评价常用的方法主要集中在化学成分上。中药制剂的生物成分分析也很重要，因为中药制剂通常既含有植物成分，也含有动物成分，其中往往包括一些错误鉴定的草药材料、掺杂物，甚至一些生物污染物。对于生物成分分析，DNA 提取的效率是一个重要因素，它可能会影响鉴定的准确性和可靠性。中药制剂的成分复杂，经过一系列处理程序后，DNA 可能会被不同程度地破坏或降解。因此，有必要建立一种从中药制剂中提取 DNA 的有效方法。在这项研究中，我们选择了一种经典的中药制剂——六味地黄丸（LDW），作为一个例子来开发一种特定于中药的 DNA 提取方法。我们对经典的十六烷基三甲基溴化铵（CTAB）法（TCM-CTAB）和三种常用的提取试剂盒进行了测试，以提取 LDW 样品中的 DNA。实验结果表明，使用 TCM-CTAB 法可获得纯度和浓度最高的 DNA。为了进一步评估不同的提取方法，我们对第二内转录间隔区（ITS2）和叶绿体基因组 trnL 内含子进行了扩增。结果表明，只有使用 TCM-CTAB 法提取的 DNA 模板才能成功进行 PCR 扩增。此外，我们使用 TCM-CTAB 法提取的 DNA 进行了高通量 454 测序。数据分析表明，从 LDW 样品中检测到了 6 种规定物种中的 3-4 种，而检测到了多达 5 种污染物种，这表明 TCM-CTAB 法可以促进后续对中药制剂的 DNA 检测。
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