复合聚合酶链反应鉴别龟甲正品与伪品的特征  

Identification and Characteristic of Testudinis carapax et Plastrum and other Turtle Species by Multiple PCR

作　　者：邓莹[1] 李明成[2] 张丽华[1,3] 

机构地区：[1]北华大学药学院,吉林吉林132013 [2]北华大学医学检验学院,吉林吉林132013 [3]吉林市雷博科技有限公司,吉林吉林132013

出　　处：《中国药学杂志》2014年第12期1022-1026,共5页Chinese Pharmaceutical Journal

基　　金：吉林省科技发展计划项目(20090906);吉林省战略性新兴产业和高新技术产业发展项目(2013G030);吉林市科技发展项目(2013523010)

摘　　要：目的探讨复合聚合酶链反应（PCR）技术鉴别中药材龟甲真伪的分子标记特征。方法采用盐析法提取龟甲正品及其伪品线粒体DNA（MitochondrialDNA，mtDNA）。从GenBank数据库中下载乌龟mtDNA细胞色素b（cytochromeb，Cytb）和细胞色素C氧化酶亚基I（cytochromeoxidasesubunit I，CoD的基因序列，用Premier5．0设计2对特异性引物，建立复合PCR技术对龟甲正品及其伪品进行扩增并测序。结果盐析法提取的龟甲mtDNA的大小均为16．6×10。bp，正品龟甲经复合PCR扩增在300～500bp间有2条明亮分明的条带，而伪品龟甲只出现1条或无条带。结论复合PCR技术鉴别龟甲真伪具有高度特异性、简便、准确、快捷、实用性强，对龟甲类药材的鉴定有很高的应用价值及准确性。，OBJECTIVE To explore the characteristic of Chinese herbal medicine Testudinis carapax et Plastrum by the multiple PCR technique. METHODS The salting out method was modified to extract mitochondrial DNA （mtDNA） from Testudinis carapax et Plastrum and their counterfeits. The sequences of cytochrome b （Cyt b） and cytochrome c oxidase subunit I （Col） genes were down- loaded from the GenBank database, and two specific pairs of primers were designed with Premier 5.0. The genes of Testudinis carapax et Plastrum were amplified with multiple PCR and sequenced. RESULTS The size of mtDNA got by salting out method was 16. 6 ~ 103 bp, and two bright stripes between 300 -500 bp were shown in agarose gel electrophoresis of authentic Testudinis carapax et Plas- trum, only one or no stripe appeared for the counterfeits. CONCLUSION The multiple PCR technique is specific, simple, and ac- curate for differentiation of authentic Testudinis carapax et Plastrum from their counterfeits.

关 键 词：龟甲 复合PCR 细胞色素C氧化酶亚基I(CoI) 细胞色素B 

分 类 号：R284[医药卫生—中药学]