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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 establish a degenerate primer PCR method for screening the first, second and third integrase genes (intⅠ), and to screen and classify the clinical isolates. Methods The optimum annealing temperature of PCR reaction was determined by gradient PCR. The PCR products were amplified by PCR from 16 clinical samples. The integration of the PCR products was analyzed by restriction endonuclease analysis. Results According to the results of gradient PCR, the optimum annealing temperature was 46 ℃. Sixteen clinical isolates were detected by PCR using degenerate primers, of which 8 were positive. Four isolates contained only the first type of integrase gene after restriction analysis, three of which contained the second type of integrase gene and one strain of integrase Contains both Class I and Class II integrase genes. Conclusion A method of simultaneous screening of the first, second and third types of integron has been established. The practical detection shows its feasibility and provides a quick and easy method for comprehensively and carefully studying the integration of sub-mediated bacterial resistance.