蒙成藥中“地格達(dá)”類原料藥材的位點特異性PCR鑒別研究

　　[摘要] 該文選擇8種含有地格達(dá)類蒙藥原料藥材的蒙成藥為對象，首先對基原植物肋柱花和苦地丁樣品采用改良CTAB法提取其總DNA，使用psbA-trnH片段進(jìn)行擴(kuò)增、測序，與GenBank下載的8種蒙成藥中其他原料藥材psbA-trnH序列進(jìn)行同源比對后根據(jù)其變異位點設(shè)計特異性鑒別引物。同時對8種蒙成藥進(jìn)行總DNA的提取，并使用DNA純化試劑盒進(jìn)行純化，通過PCR反應(yīng)對葉綠體rbcL序列進(jìn)行擴(kuò)增，再分別選擇肋柱花和苦地丁鑒別引物進(jìn)行擴(kuò)增，并將擴(kuò)增后的產(chǎn)物進(jìn)行測序。所得序列校正、比對后，進(jìn)行比對分析。結(jié)果表明，地格達(dá)-4湯、地格達(dá)-8散、地格達(dá)-4散中均含有原料藥材肋柱花，苦地丁特異鑒別引物擴(kuò)增分析可以鑒定出利膽八味散、伊赫哈日-12和阿嘎日-35中含有苦地丁。該研究結(jié)果說明，位點特異PCR方法用于鑒定蒙成藥中原料藥材具有一定的可行性。
　　[關(guān)鍵詞] 分子鑒別;蒙成藥;“地格達(dá)”;位點特異性PCR
　　[收稿日期] 2014-11-16
　　[基金項目] 國家自然科學(xué)基金項目（81060372）;國家“十二五”科技支撐計劃項目 （2012BAI28B02）;中醫(yī)藥行業(yè)專項（201407003）;內(nèi)蒙古自治區(qū)高等學(xué)�？茖W(xué)研究項目（NJZY12222）
　　[通信作者] 李旻輝，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

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