三类整合酶基因(intⅠ)的简并引物PCR方法建立及应用  

Construction and application of degenerate primers PCR assay to screen class 1, 2 and 3 integrase (intⅠ)

作　　者：李心晖[1] 石磊[1] 杨维青[2] 曹以诚[1] 张宏梅[1] 尹晓琳[2] 郑美萍[1] 

机构地区：[1]华南理工大学食品与生物工程学院,广州510640 [2]河北医科大学基础医学院微生物和免疫教研室

出　　处：《中华微生物学和免疫学杂志》2005年第2期156-160,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家教育部留学回国人员科研启动金资助项目 (B7 3 2 1 0 90 ) ;华南理工大学高水平大学建设项目资助 (B12 3 49)

摘　　要：目的　建立一种筛选第一、二和三类整合酶基因 (intⅠ )的简并引物PCR方法 ,并对临床菌株进行整合子筛选和分类。方法　用梯度PCR方法确定PCR反应的最适退火温度 ,并对 1 6株临床标本进行PCR扩增 ,通过对阳性PCR扩增产物的限制性内切酶分析进行整合子分类。结果　根据梯度PCR结果确定最适退火温度为 4 6℃。应用简并引物PCR法检测 1 6株临床分离株 ,其中 8株阳性 ,经酶切分类后确定其中 4株只含有第一类整合酶基因 ,有 3株含有第二类整合酶基因 ,1株同时含有第一类和第二类整合酶基因。结论　建立了一种同时筛选第一、二和三类整合子的方法 ,实际检测显示其可行性 ,为全面细致研究整合子介导的细菌耐药提供了一种快速、简便的方法。Objective To construct a PCR assay using degenerate primers to screen class 1,2 and 3 integrase (intⅠ). Methods Degenerate primers were designed and then gradient PCR was used to decide the optimization annealing temperature. The PCR amplicons were analyzed by digestion with restriction endonucleases. Results 46℃ of optimization annealing temperature was decided according to the gradient PCR results. 16 of clinical strains were used in this study, 8 of strains were positive for PCR. The amplicons digested with restriction endonucleases showed that 4 of strains harbored intⅠ1, 3 of strains harbored intⅠ2, 1 of strains harbored both intⅠ1 and intⅠ2. Conclusion A PCR assay to screen class 1, 2 and 3 integron at one time was established and confirmed to be practicable by application. A swift and versatile method is provided for comprehensive and particular research of antibiotic resistance genes carried by integrons in bacteria.

关 键 词：简并引物PCR 酶基因 方法建立 限制性内切酶分析 PCR扩增产物 PCR方法 PCR法检测 退火温度 PCR反应 梯度PCR 临床分离株 整合子 临床菌株 临床标本 细菌耐药 筛选 阳性 

分 类 号：R346[医药卫生—基础医学]