蒙成药中“地格达”类原料药材的位点特异性PCR鉴别研究

　　[摘要] 该文选择8种含有地格达类蒙药原料药材的蒙成药为对象，首先对基原植物肋柱花和苦地丁样品采用改良CTAB法提取其总DNA，使用psbA-trnH片段进行扩增、测序，与GenBank下载的8种蒙成药中其他原料药材psbA-trnH序列进行同源比对后根据其变异位点设计特异性鉴别引物。同时对8种蒙成药进行总DNA的提取，并使用DNA纯化试剂盒进行纯化，通过PCR反应对叶绿体rbcL序列进行扩增，再分别选择肋柱花和苦地丁鉴别引物进行扩增，并将扩增后的产物进行测序。所得序列校正、比对后，进行比对分析。结果表明，地格达-4汤、地格达-8散、地格达-4散中均含有原料药材肋柱花，苦地丁特异鉴别引物扩增分析可以鉴定出利胆八味散、伊赫哈日-12和阿嘎日-35中含有苦地丁。该研究结果说明，位点特异PCR方法用于鉴定蒙成药中原料药材具有一定的可行性。
　　[关键词] 分子鉴别;蒙成药;“地格达”;位点特异性PCR
　　[收稿日期] 2014-11-16
　　[基金项目] 国家自然科学基金项目（81060372）;国家“十二五”科技支撑计划项目 （2012BAI28B02）;中医药行业专项（201407003）;内蒙古自治区高等学校科学研究项目（NJZY12222）
　　[通信作者] 李旻辉，E-mail： li_minhui@aliyun.com
　　Study on identification of "Digeda" raw materials in Mongolian patent
　　medicine by PCR amplification of specific alleles
　　CUI Zhan-hu， HUANG Xian-zhang， LONG Ping， ZHANG Le， ZHAO Dong-dong， WANG Ying-li， LI Min-hui
　　（1.Nanyang Institute of Technology， Nanyang 473004， China;
　　2. State Key Laboratory of Dao-di Herbs Breeding Base， National Resource Center for Chinese Materia Medica，
　　China Academy of Chinese Medical Sciences， Beijing 100700， China;
　　3. Baotou Medical College， Baotou 014060， China;
　　4. Inner Mongolia Autonomous Region Academy of Chinese Medicine， Huhhot 010020， China）
　　[Abstract] To explore a new method for identification of Mongolian patent medicine （MPM） by PCR amplification of specific alleles. Eight kinds of MPM were used to study the identification of ″Digeda″ raw materials. The total DNA of Lomatogonium rotatum and Corydalis bungeana samples were extracted through modified CTAB method， psbA-trnH sequence was amplified by PCR and sequenced directionally. Specific primer was designed. The DNA of 8 kinds of MPM also was extracted and purified by the commercial DNA purification kits. The rbcL and two pair of specific primers sequences were amplified. The specific amplified products were sequenced in forward directions. All specific sequences were aligned and were analyzed. The results indicated that L. rotatum can be identified by specific primers from Digeda-4 Tang， Digeda-8 San， Digeda-4 San， and C. bungeana medicinal materials can be identified by specific primers from Li Dan Ba Wei San， Yi He Ha Ri-12 and A Ga Ri-35. PCR amplification of specific alleles can stably and accurately distinguish raw medicinal materials in MPM.
　　[Key words] molecular identification; Mongolian patent medicine; ″Digeda″ medicinal plants; PCR amplification of specific allele
　　doi：10.4268/cjcmm20150504

相关热词搜索：成药 特异性 鉴别 位点 格达