中国中药杂志

2014, v.39(19) 3684-3688

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基于ITS序列位点特异性PCR的肉苁蓉与其混伪品的鉴别研究
Study on identification of Cistanche Hebra and its adulterants by PCR amplification of specific alleles based on ITS sequences

李振华;龙平;邹德志;李越;崔占虎;李旻辉;
LI Zhen-hua;LONG Ping;ZOU De-zhi;LI Yue;CUI Zhan-hu;LI Min-hui;Baotou Medical College;State Key Laboratory of Dao-di Herbs,Chinese Materia Medica Resources Center,China Academy of Chinese Medicinal Sciences;

摘要(Abstract):

研究荒漠肉苁蓉、锁阳和黄花列当之间的DNA分子鉴别方法,采集不同产地的30份荒漠肉苁蓉,13份锁阳以及8份黄花列当,所有样品进行总DNA提取,通过对其ITS片段进行扩增,测序。再进行同源比对后根据其变异位点设计特异性鉴别引物,并对位点特异性PCR的反应条件进行了优化;另外,对位点特异性PCR法与DNA序列分析法的鉴别进行了比较。结果显示,该研究设计的位点特异引物在同一PCR反应中,荒漠肉苁蓉能扩增出331 bp的条带,而混伪品锁阳和黄花列当不能扩增出条带,从而实现了正伪品的鉴别。该文通过位点特异PCR的方法可以实现荒漠肉苁蓉与混伪品锁阳、黄花列当的快速、准确鉴别。
To explore the new method of discriminating Cistanche deserticola,Cynomorium songaricum and Orobanche pycnostachya by using PCR amplification of specific alleles. 30 samples of the different C. deserticola,21 samples of C. songaricum and O.pycnostachya were collected. The total DNA of the samples were extracted,the ITS sequences from C. deserticola,C. songaricum and O. pycnostachya were amplified by PCR and sequenced unidirectionally. These sequences were aligned by using Clustul W. Specific primer was designed according to the ITS sequences of specific alleles,and PCR reaction system was optimized. Additionally,compare with the identification of specific PCR method and DNA sequence analysis method. The result showed that the 331 bp identification band for C. deserticola and the adulterants not amplified bands by a single PCR reaction,which showed good identification ability to the three species. PCR amplification of specific alleles can be used to identify C. deserticola,C. songaricum and O. pycnostachya successfully.

关键词(KeyWords): 荒漠肉苁蓉;位点特异性PCR;分子鉴定
Cistanche deserticola;PCR amplification of specific alleles;molecular identification

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基金项目(Foundation): 国家“十二五”科技支撑计划项目(2012BAI28B02);; 中央本级重大增减支项目(20603020116,20603020220);; 中医药行业科研专项(201407003);; 科技基础性工作专项重点项目(2007FY110600)

作者(Author): 李振华;龙平;邹德志;李越;崔占虎;李旻辉;
LI Zhen-hua;LONG Ping;ZOU De-zhi;LI Yue;CUI Zhan-hu;LI Min-hui;Baotou Medical College;State Key Laboratory of Dao-di Herbs,Chinese Materia Medica Resources Center,China Academy of Chinese Medicinal Sciences;

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