细菌耐药整合子intI分类的多重PCR方法的建立及应用

目的建立快速筛选细菌耐药整合子的分类方法,并对21株来自临床的菌株进行整合子筛选和分类。方法根据G enB ank/EM BL内的第一、第二和第三类的整合酶序列,通过软件C lustalW的多重比对分析设计出各类整合子的特异性引物,用对照菌株建立多重PCR方法并对21株临床菌株进行PCR扩增,通过其PCR扩增产物片段大小的不同进行整合子分类。结果对照菌株实验结果显示,21株临床分离菌株中,15株在565bp处有扩增片段,即为含有第一类整合酶基因;4株在403bp处有扩增片段,即含有第二类整合酶基因;1株在565bp和403bp处均有扩增片段,提示其同时含有第一、二类整合酶基因;1株没有得到任何扩增片段,即不含有这三类整合酶基因;在被测菌株中未发现第三类整合酶基因的阳性菌株。结论本文报道的筛选细菌耐药整合酶基因分类的多重PCR方法效果良好,具有可行性,为更加全面细致研究整合子类型以及整合子介导的细菌耐药机制提供了一种简单易行、快捷有效的方法。

Construction and application of multi-PCR assay to screen antibiotic resistance integrons in bacteria

Objective To establish and evaluate a multi-PCR assay for screening class 1,2 and 3 integrases gene(intI).Methods Specific primers of intI1,intI2 and intI3 were designed according to the multi-aligment result and the multi-PCR assay was used to classify the integrons.Results Twenty-one of clinical strains were used in this study;15 strains presented a 546bp amplicon and it was shown that these strains harbored intI1;4 strains presented a 403bp amplicon and it was shown these strains harbored intI2;1 strain presents both a 565bp and a 403bp amplicon and this strain harbored both intI1 and intI2;1 strain was negative for multi-PCR.None of intI3 genes were found in these strains.Conclusion The multi-PCR assay to screen class 1,2 and 3 integrons at one time was constructed and confirmed to be practicable by application.It also provides a swift and versatile method for comprehensive and particular research of antibiotic resistance genes carried by integrons in bacteria.