运用变性高效液相色谱快速检测CTX-M超广谱β内酰胺酶基因型

Rapid detection of the genotype of CTX-M-type extended spectrum beta-lactamase by denaturing high-performance liquid chromatography

目的直用变性高效液相色谱(DHPLC)技术对确认产 ESBLs 的大肠埃希菌和肺炎克雷伯菌临床分离株 CTX-M 型质粒酶进行基因分型,探讨其灵敏度和特异度,以建立快速检测 CTX-M 型 ESBLs 的新方法。方法采用 PCR 技术从大肠埃希菌和肺炎克雷伯菌临床分离株中扩增出 CTX-M 型质粒的编码序列,用 DHPLC 技术对扩增产物进行分析和 DNA 测序,确定其基因突变的类型,通过比对两者结果后确定其基因型。结果从34株产 ESBLs 大肠埃希菌和肺炎克雷伯菌临床分离株中扩增出35份 CTX-M 型 ESBLs 编码序列(其中1株同时扩增到2份 CTX-M 型编码序列)。11份与标准菌株 CTX-M-3一致:4份与标准菌株 CTX-M-9一致;测序确定分别为 CTX-M-3型和 CTX-M-9型:20份表现为3种形态各异的异常洗脱峰,测序结果表明20份样本与其标准株对比均存在着基因突变,DHPLC 中异常峰一致的样本均为同一基因亚型。结论 DH-PLC 可用于 ESBLs 基因分型的检测,能快速检测 CTX-M 的基因型,具有准确性高、简便快捷、经济等特点,有很大的临床应用价值。

Objective To determine the prevalence and genotypes of clinical Klebsiella pneumoniae and Escherichia coli producing CTX-M-type ESBLs by denaturing high performance liquid chromatography （DHPLC） in Guangzhou area; evaluate the sensitivity and specificity of DHPLC to establish a rapid and convenient method for detecting the genotype of CTX-M type ESBLs, Methods Genotype determination of CTX-M-type ESBLs was performed by PCR amplification followed by DHPLC and DNA sequencing. Results Thirty-five amplicons were amplified from 34 isolates of K. pneumoniae and E. coli, including one strain producing two types of CTX-M-type ESBLs, Morphology of eleven strains was identical to that of CTX-M 3 and morphology of four strains was that of CTX-M-9 by DHPLC, By sequencing analysis, they were characterized as CTX M-3 and CTX-M 9 respectively, Three abnormal elution peaks were found in 20 amplicons which had DNA mutations and each genotype corresponded to specific peak pattern. Both the sensitivity and specificity of DHPLC were 100% in this study, Conclusions DHPLC can be used to detect the genotype of CTX-M-type ESBLs. DHPLC is highly accurate, convenient and rapid technique for detecting genotype and has good promise for clinical use.