DNA extraction protocol for biological ingredient analysis of Liuwei Dihuang Wan.
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.
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.
Xinwei
X
Cheng
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.
Xiaohua
X
Chen
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.
Xiaoquan
X
Su
Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan 250062, China.
Huanxin
H
Zhao
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.
Maozhen
M
Han
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.
Cunpei
C
Bo
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.
Jian
J
Xu
Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan 250062, China. Electronic address: baihong@gmail.com.
Hong
H
Bai
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. Electronic address: ningkang@qibebt.ac.cn.
Kang
K
Ning
D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D004247:DNA / Q000302:isolation & purification*; D021901:DNA, Intergenic / Q000235:genetics; D004365:Drugs, Chinese Herbal / Q000737:chemistry*; D006801:Humans; D059627:Liquid Phase Microextraction / Q000379:methods*; D010940:Plant Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012997:Solvents
D002593:Cetrimonium Compounds; D021901:DNA, Intergenic; D004365:Drugs, Chinese Herbal; D010940:Plant Proteins; D012997:Solvents; C476907:liu-wei-di-huang wan; D004247:DNA; D000077286:Cetrimonium
Biological ingredients; DNA extraction; Liuwei Dihuang Wan; Traditional Chinese medicine preparation