反向PCR扩增Ⅰ类整合酶基因侧翼序列.docVIP

 反向 PCR 扩增类整合酶基因侧翼序列# 陈体，冯银，袁金玲，漆涌，伍勇** （中南大学湘雅三医院检验科，长沙 410013） 5 10 摘要：目的：采用反向 PCR 对类整合酶基因侧翼序列进行检测分析。方法： 以临床分离 的类整合子阳性大肠埃希菌为研究对象，用限制性内切酶 Hind 对基因组 DNA 进行酶 切，T4 DNA 连接酶使酶切产物自身环化，设计类整合酶基因反向引物扩增类整合酶基 因侧翼序列。结果： 临床分离大肠埃希菌中类整合子检出率为 68.1%，反向 PCR 扩增得 到 1800bp 大小的类整合酶基因侧翼序列。结论：类整合子在临床分离大肠埃希菌中广 泛存在，反向 PCR 对类整合酶基因侧翼序列检测进一步明确了类整合子的结构，并发 现其侧翼序列存在 RNA 聚合酶基因β亚基，为类整合子调控基因分析及克隆奠定了研究 基础。 关键词：临床检验诊断学；反向 PCR；整合子；整合酶；侧翼序列 中图分类号：R446.5 15 Using reverse PCR to analysis flanking DNA regions of class 1 integrase CHEN Ti, FENG Yin, YUAN Jinling, QI Yong, WU Yong (Department of Clinic Laboratory, The Third Xiangya Hospital of Central South University, 20 25 30 35 40 Changsha 410013) Abstract: Objective: The flanking DNA regions of class 1 integrase 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 Ⅰintegrase gene. Results: Study of clinic samples indicated that classⅠ integron was expressed in 68.1% of Escherichia coli strains. 1800bp DNA fragment can be obtained from flanking DNA regions of class 1 integrons by reverse PCR. Conclusion: Our clinic study indicated that distribution of class Ⅰintegron in Escherichia coli is prevalent. And we further identified the structure of classⅠ integron, there is RNA polymerase gene β subunits in flanking region of classⅠ integron. Our researches provide support for the gene analysis of the regulation of class Ⅰintegron and cloning research in the future. Key words: Clinical Laboratory Diagnostics; reverse PCR; integron; integrase; flanking DNA regions 整合子是细菌尤其是革兰阴性菌多重耐药快速发展的重要原因之一，在整合酶的作用下 能够识别并俘获或删除移动性基因盒，基因盒可在位于整合子上游的启动子的作用下得以表 达，使得耐药基因在不同细菌种、属间进行播散[1]。近年来整合子系统对细菌耐药形成的作 用备受关注[2-5]，整合子根据整合酶基因序列的不同主要有 4 类，其中 I 类整合子在临床多 重耐药的细菌中最为常见[6]。整合子可携带多种耐药基因盒，包括氨基糖苷类、喹诺酮类和 磺胺类等耐药基因，作为一种天然克隆和表达载体，整合子介导的耐药