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

目的应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-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。

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

Objective To determine the prevalence and genotypes of clinical Klebsiella pneumoniae and Escherichia coli produ- cing CTX-M-type ESBLs by denaturing high-performance liquid chromatography(DHPLC)in Guangzbou area;evaluate the sensitivity and specificity of DHPLC to establish a rapid and convenient method for detecting the genotype of CTX-M-type ES- BLs.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 tbat of CTX-M-3 and mor- phology 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.