金钱白花蛇快速PCR鉴别方法的建立  

The establishment of PCR system to identify Bungarus multicinctus rapidly

作　　者：赵静雪[1,2] 崔光红[2] 辛敏通[3] 唐仕欢[2] 

机构地区：[1]首都医科大学中医药学院,北京100069 [2]中国中医科学院中药研究所,北京100700 [3]北京市药品检验所,北京100035

出　　处：《药学学报》2010年第10期1327-1332,共6页Acta Pharmaceutica Sinica

基　　金：国家重点基础研究发展计划(2006CB504700);<中国药典>2010版一部标准研究项目(YD-195);中国中医科学院自主选题项目(02085)

摘　　要：本文旨在建立一种快速、高效的鉴别金钱白花蛇药材及其伪品的PCR方法。通过对金钱白花蛇及其伪品的Cytb序列进行序列比对分析,设计1对能准确鉴别正品金钱白花蛇和其伪品的引物。采用商品化基因组DNA提取试剂盒提取基因组总DNA,采用两步循环PCR程序进行PCR鉴别,并对影响PCR结果的主要因素(退火温度、Taq酶用量、循环次数等)进行方法学考察和优化,对不同公司PCR仪、Taq酶产品进行普适性试验。在方法学考察的基础上,以试剂盒提取药材正品13批、伪品20批,反应参数:95℃预变性5min,循环反应30次(95℃30s,55℃45s),72℃延伸5min的条件可使所有受检样品得到准确鉴定,鉴别过程可在4h内完成,适合于金钱白花蛇PCR鉴别方法的推广应用。The purpose of the present study is to establish a rapid and effective PCR method for the identification of B. multicinctus. Based on sequence alignment of B. multicinctus and its adulterants, we found that Cyt b gene is a good molecular genetic marker for the authentication of B. multicinctus. On the basis of the sequence data, a pair of highly specialized primers was designed. The templates were extracted by the DNA purification system. Key factors such as annealing temperature, concentration of Taq enzyme and cycle numbers were analyzed and optimized. The modified PCR program consisted of an initial denaturation step at 95 ℃ for 5 min, followed by 30 cycles of 95 ℃ for 30 s and 55 ℃ for 45 s and a final extension at 72 ℃ for 5 min. Thirteen samples of B. multicinctus were identified accurately from their 20 adulterants in 4 hours. The results indicated it is a highly accurate, rapid and applicable method for the authentication of B. multicinctus.

关 键 词：金钱白花蛇 PCR 鉴别 

分 类 号：R931[医药卫生—生药学]