Chao Zhi, Liao Jing, Liang Zhenbiao, Huang Suhua, Zhang Liang, Li Junde
Department of Pharmacognosy, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China.
Department of Pharmacognosy, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China ; Beijing Royal Intergrative Medicine Hospital, Beijing, China.
Pharmacogn Mag. 2014 Oct;10(40):449-57. doi: 10.4103/0973-1296.141816.
To test the feasibility of DNA barcoding for accurate identification of Jinqian Baihua She and its adulterants.
Standard cytochrome C oxidase subunit I (COI) gene fragments were sequenced for DNA barcoding of 39 samples from 9 snake species, including Bungarus multicinctus, the officially recognized origin animal by Chinese Pharmacopoeia, and other 8 adulterate species. The aligned sequences, 658 base pairs in length, were analyzed for divergence using the Kimura-2-parameter (K2P) distance model with MEGA5.0.
The mean intraspecific K2P distance was 0.0103 and the average interspecific genetic distance was 0.2178 in B. multicinctus, far greater than the minimal interspecific genetic distance of 0.027 recommended for species identification. A neighbor-joining (NJ) tree was constructed, in which each species formed a monophyletic clade with bootstrap supports of 100%. All the data were submitted to Barcode of Life Data system version 3.0 (BOLD, http://www.barcodinglife.org) under the project title "DNA barcoding Bungarus multicinctus and its adulterants". Ten samples of commercially available crude drugs of JBS were identified using the identification engine provided by BOLD. All the samples were clearly identified at the species level, among which five were found to be the adulterants and identified as Dinodon rufozonatum.
DNA barcoding using the standard COI gene fragments provides an effective and accurate means for JBS identification and authentication.
测试DNA条形码技术用于准确鉴定金钱白花蛇及其伪品的可行性。
对9种蛇类的39个样本进行标准细胞色素C氧化酶亚基I（COI）基因片段测序，用于DNA条形码分析，其中包括《中国药典》认可的正品来源动物多环扁尾海蛇，以及其他8种伪品。使用MEGA5.0软件，采用Kimura-2-参数（K2P）距离模型，对长度为658个碱基对的比对序列进行分歧分析。
多环扁尾海蛇种内平均K2P距离为0.0103，种间平均遗传距离为0.2178，远大于物种鉴定推荐的最小种间遗传距离0.027。构建了邻接（NJ）树，每个物种形成一个单系分支，自展支持率为100%。所有数据已提交至生命条形码数据系统3.0版（BOLD，http://www.barcodinglife.org），项目名称为“多环扁尾海蛇及其伪品的DNA条形码”。使用BOLD提供的鉴定引擎对10个市售金钱白花蛇药材样本进行鉴定。所有样本均在物种水平上得到明确鉴定，其中5个被鉴定为伪品，为赤链蛇。
使用标准COI基因片段的DNA条形码技术为金钱白花蛇的鉴定和认证提供了一种有效且准确的方法。
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