变性高效液相色谱技术检测qnr阳性肺炎克雷伯菌染色体突变位点

Detecting point mutations of qnr positive Klebsiella pneumoniae by denaturing high-performance liquid chromatography

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 point mutations at GyrA and topoisomerase ParC of qnr positive Klebsiella pneumoniae.Methods Denaturing high-performance liquid chromatography（DHPLC） and DNA sequencing technique were used for the detection.Results There were four kinds of point mutations in GyrA and two kinds of point mutations in ParC in qnr positive Klebsiella pneumoniae strains.Conclusion Point mutations in GyrA and ParC are related to different quinolone resistance levels of clinical isolated qnr positive Klebsiella pneumoniae.They play an important part in highly quinolone resistant strains.DHPLC coupled with DNA sequencing are efficient methods for the detection of small fragment DNA mutations.