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目的检测质粒介导喹诺酮耐药基因(quinolone resistance,qnr)阳性肺炎克雷伯菌(Klebsiella pneumoniae,Kpn)染色体旋转酶GyrA和拓扑异构酶ParC的突变位点。方法应用变性高效液相色谱技术(DHPLC)对有异常峰型的菌株进行DNA测序。结果 qnr阳性Kpn GyrA亚基存在4种氨基酸突变类型,ParC亚基存在2种氨基酸突变类型。结论喹诺酮作用靶位GyrA和ParC亚基是否存在氨基酸变异与临床qnr阳性菌株耐药水平的差异有关,其位点突变对高水平耐药起重要作用。DHPLC联合测序可用于高通量检测微生物小片段基因点突变。
Objective To detect the mutation sites of plasmid - mediated gyrA and topoisomerase ParC of quinolone resistance (qnr) - positive Klebsiella pneumoniae (Kpn). Methods Denaturing high performance liquid chromatography (DHPLC) was used to perform DNA sequencing on strains with abnormal peak shape. Results There were four kinds of amino acid mutations in qnr-positive Kpn GyrA subunit, and two kinds of amino acid mutations in ParC subunit. Conclusions The amino acid variation of GyrA and ParC subunits of quinolone target is related to the difference of drug resistance of clinical qnr positive strains. The mutation of the site plays an important role in high level drug resistance. DHPLC co-sequencing can be used for high-throughput detection of microbe fragments gene point mutations.