基于分子生物学特异性扩增的方法鉴别菲牛蛭  

On the Identification Method of Poecilobdella manillensis Based on Molecular Biological Specific Amplification

作　　者：过立农[1] 刘杰[1,2] 杨宝[3] 昝珂 郑健[1] 马双成[1] 马宁[3] 李昀铮 Guo Linong;Liu Jie;Yang Bao;Zan Ke;Zheng Jian;Ma Shuangcheng;Ma Ning;Li Yunzheng(National Institutes for Food and Drug Control,Beijing 100050,China;Minzu University of China,Beijing 100081,China;Taiyuan Institute for Food and Drug Control,Taiyuan 030000,China;Songyuan Institute for Food and Drug Control,Songyuan 138000,China)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]中央民族大学,北京100081 [3]太原市食品药品检验所,太原030000 [4]松原市食品药品检验所,松原138000

出　　处：《中国药事》2018年第9期1239-1244,共6页Chinese Pharmaceutical Affairs

基　　金：中药饮片质量保障体系研究(一)(编号201507002)

摘　　要：目的:通过DNA条形码鉴定和建立特异性引物扩增的方法,有效区分菲牛蛭与其他水蛭品种。方法:通过聚合酶链式反应(PCR)对水蛭样品的COI序列进行扩增,利用MEGA 6.06软件通过建树法进行NJ树聚类分析,针对菲牛蛭素的编码基因序列、利用Primer Premier 5软件进行引物设计,对经设计、合成的8对引物通过PCR扩增和琼脂糖凝胶电泳实验进行筛选,并确定特异性引物的最佳退火温度。结果:通过COI序列的NJ树聚类分析可以确定20批水蛭样品中的9批菲牛蛭样品,经过Primer Premier 5软件进行引物设计共选出8对引物进行合成,通过PCR扩增和琼脂糖凝胶电泳预实验筛选出引物7、为最佳实验引物,且确定了引物7的最佳退火温度为49℃。结论:通过COI序列的DNA条形码研究,确定了样品中的菲牛蛭,通过特异性引物PCR扩增和电泳检测,有效地将菲牛蛭与其他水蛭样品进行区分,为菲牛蛭的分子生物学鉴定方法提供了参考。Objective： To identify Poecilobdella manillensis from other Hirudos through DNA barcoding method and specific primers. Methods： COI sequences of Poecilobdella manillensis were amplified with polymerase chain reaction（PCR）, through the method of Neighbour Joining to draw the spectral of clustering analysis tree using the software of MEGA 6.06, primers were designed by using the software of Primer Premier 5 based on the coding gene sequence of bufudin, the best one was screened from 8 designed primers through the PCR and agarose gel electrophoresis（AGE） experiment, and the optimum annealing temperature of specific primers was determine. Results： The Neighbour Joining clustering analysis of COI sequences can effectively distinguish the 9 lots of Poecilobdella manillensis among the 20 samples for this study, the PCR and AGE experiments confirmed primers-7 as the best primer, and the optimum annealing temperature was 49℃. Conlusions： Through the DNA Barcoding study on the COI sequence Poecilobdella manillensis was identified from the 20 lots of samples, the specific PCR and AGE experiment on primers-7 can effectively identify Poecilobdella manillensis from other Hirudos, which can provide the reference basis for molecular biology identification techniques of Poecilobdella manillensis and the identification of other traditional medicines.

关 键 词：菲牛蛭 聚合酶链式反应(PCR) COⅠ DNA条形码 特异性扩增 

分 类 号：R917[医药卫生—药物分析学]