摘要
目的 建立一种简便、准确的鹿类中药材鹿茸、鹿鞭、鹿筋、鹿胎的DNA分子标记鉴定方法。方法 在对鹿类中药材的正品原动物梅花鹿、马鹿及其混伪品原动物的Cytb基因全序列分析的基础上 ,设计了一对专用于鉴定正品鹿类药材的位点特异性鉴别引物ILu0 1 L和ILu0 1 H。结果 在 6 4℃的复性温度下 ,用鉴别引物对原动物样品进行鉴别PCR ,仅正品能得到约 36 5bp阳性扩增带 ;对鹿茸、鹿鞭及鹿筋正、伪品药材进行PCR鉴定 ,结果表明 :3批鹿茸仅一批为正品 ,2批鹿鞭皆为伪品 ,鹿筋正、伪品药材PCR鉴定与形态鉴定结果一致。随机选取 2枚鹿茸及一个原动物做Cytb基因片段序列分析 ,其结果与PCR鉴定完全一致。结论 对市售鹿类商品药材需加强质量监督和管理。所设计的鉴别引物对梅花鹿、马鹿有高度特异性 ,可应用于以其为原动物的鹿类中药材的鉴定。
AIM To develop a convenient and accurate method of DNA molecular marker for the identification of traditional Chinese medicines made of deers, consisting of pilose antler, penis and testis, tendon and foetus. METHODS Based on the analysis of DNA sequence of mitochondrial Cyt b gene from original animals of both genuine crude drugs, Cervus nippon and Cervus elaphus , and adulterants, a pair of allele specific primers named as ILu01 L and ILu01 H were designed for distinguishing geniune crude drugs of deers from their adulterants. RESULTS The results of diagnostic PCR annealing at 64℃ for original animals showed that a 365 bp fragment was only amplified from DNA templates of Cervus nippon and Cervus elaphus. For the identification of medicinal materials total of 43 samples from 6 packages were tested under the same reaction conditions except for DNA templates extracted from these crude drugs. Only 9 samples mentioned above was shown to generate positive amplificon. The result indicate that of 8 samples from 1 package of pilose antler and only 1 sample of deer tendon was genuine crude drug. After that, 3 amplified fragments selected randomly were performed with sequencing analysis with the purpose of verifying the results from diagnostic PCR. Data from sequencing confirmed the reliability of diagnostic PCR identification. CONCLUSION The diagnostic primers designed in the present study were highly specific for Cervus nippon and Cervus elaphus , and they could be used for the authentication of traditional Chinese medicines made from the deer. The quality of the crude drugs of the deer in the current market is a problem and more effective quality control for these traditional Chinese medicines is urgently needed.
出处
《药学学报》
CAS
CSCD
北大核心
2001年第8期631-635,共5页
Acta Pharmaceutica Sinica
基金
国家自然科学基金 (39870 913)
江苏教委科学基金(98KJB36 0 0 10 )
江苏省"333跨世纪学术带头人培养工程"基金资助
关键词
鹿类中药材
CYT
B基因
鉴别引物
PCR鉴定
traditional Chinese medicines made of deers
Cytb gene
diagnostic primers
PCR identification
