反向PCR扩增Ⅰ类整合酶基因侧翼序列  

Using reverse PCR to analysis flanking DNA regions of class Ⅰ integrons

作　　者：冯银[1] 袁金玲[1] 伍勇[1] 漆涌[1] 陈体[1] 

机构地区：[1]中南大学湘雅三医院检验科,湖南长沙410013

出　　处：《国际检验医学杂志》2013年第8期919-920,共2页International Journal of Laboratory Medicine

基　　金：湖南省自然科学基金资助项目(08JJ3069);湖南省科技厅计划资助项目(2010FJ3079)

摘　　要：目的采用反向PCR对Ⅰ类整合酶基因侧翼序列进行检测分析。方法以临床分离的Ⅰ类整合子阳性大肠埃希菌为研究对象,用限制性内切酶HindⅢ对基因组DNA进行酶切,T4 DNA连接酶使酶切产物自身环化,设计Ⅰ类整合酶基因反向引物扩增Ⅰ类整合酶基因侧翼序列。结果临床分离大肠埃希菌中Ⅰ类整合子检出率为68.1%,反向PCR扩增得到1800bp大小的Ⅰ类整合酶基因侧翼序列。结论Ⅰ类整合子在临床分离大肠埃希菌中广泛存在,反向PCR对Ⅰ类整合酶基因侧翼序列检测进一步明确了Ⅰ类整合子的结构,并发现其侧翼序列存在RNA聚合酶基因β亚基,为Ⅰ类整合子调控基因分析及克隆奠定了研究基础。Objective The flanking DNA regions of class 1 integrons were detected using reverse PCR. Methods Genomic DNA of Escherichia coli strains from clinic samples were digested by the restriction enzymes Hind Ⅲ. With the help of T4 DNA ligase, the digested fragments will self-ligase. Reverse primers were designed to amplify the 5＇ and 3＇ flanking sequences of class Ⅰ inte-grase gene. Results Study of clinic samples indicated that class Ⅰ integron was expressed in 68.1% of Escherichia coli strains. 1 800 bp DNA fragment can be obtained from flanking DNA regions of class Ⅰ integrons by reverse PCR. Conclusion Our clinic study indicated that distribution of class Ⅰ integron in Escherichia coli is prevalent. And we further identified the structure of class Ⅰ inte-gron, there is RNA polymerase gene β subunits in flanking region of class Ⅰ integron. Our researches provide support for the gene a-nalysis of the regulation of class Ⅰ integron and cloning research in the future.

关 键 词：聚合酶链反应 基因 整合酶类 

分 类 号：R440[医药卫生—诊断学]