中药材龟甲的分子鉴定研究

用PCR产物直接测序法对中药材龟甲(板)进行鉴别。从乌龟 Chinemys revesii 和其他20种产地为中国或东南亚国家的龟类的组织材料中提取DNA,扩增约110bp的线粒体12SrRNA,基因片段并进行序列分析,构建了21种龟类的12SrRNA基因片段序列数据库。序列比较的结果表明乌龟与其它20种龟类的这段序列均有差别,序列差异在3.7～15.7%之间。从江苏省药品检验所提供的19块龟甲检品上各取样0.1～0.5g提取 DNA,扩增与上述相同的基因片段,与构建的数据库进行比较,结果表明19块龟甲中只有3块的原动物为乌龟,其余的龟甲均为混淆品。本文的结果为药材龟甲的鉴定找到了有效、可靠的分子遗传标记方法。

Molecular identification of the Chinese drug turtle shells]

This paper reports a new method to identify the Chinese drug turtle shells using PCR product direct sequencing method. DNA was extracted from tissues of the Chinese three-keeled pond turtle Chinemys reevesii and 20 other species of turtles occurring in China and Southeast Asian countries. One hundred and ten base pairs of mitochondrial 12S rRNA gene fragment were amplified from the extract using PCR technique and obtained sequences. These sequences were used to construct 12S rRNA gene fragment sequence database for the 21 turtle species. Comparison of these sequences indicated that the sequence from the Chinese three-keeled pond turtle is different from that of all the other 20 turtle species. The sequence divergence is 3.7-15.7%. DNA was extracted from 0.1-0.5 g of shell from 19 turtle shells provided by the Jiangsu Institute for Drug Control and 12S rRNA gene fragment was amplified and sequenced. Comparison of the sequences from the 19 turtle shells and 12S rRNA gene fragment sequence database indicated that only 3 samples are shells of the Chinese three-keeled pond turtle specified in the Phamacopoeia of the People's Republic of China and the others are substitutes. The technique used in the present paper was found to be effective and reliable for the identification of turtle shells.