变性高效液相色谱对CTX-M型超广谱β内酰胺酶基因分型研究  

Genotype of CTX-M-type extended-spectrum beta-lactamases by denaturing high-performance liquid chromatography

作　　者：段辉丽[1] 刘文恩[1] 陈腊梅[1] 李虹玲[1] 潘军[1] 邹明祥[1] 许力[2] 

机构地区：[1]中南大学湘雅医院检验科,长沙410008 [2]英国伯明翰大学医学院

出　　处：《中华检验医学杂志》2009年第6期678-682,共5页Chinese Journal of Laboratory Medicine

基　　金：湖南省卫生厅科研课题资助项目（B2004-025）

摘　　要：目的 探讨湖南省CTX-M型超广谱β内酰胺酶（ESBLs）基因型分布情况和变性高效液相色谱（DHPLC）方法检测CTX-M型ESBLs基因型的准确性。方法用多重PCR扩增标准菌株和ESBLs表型阳性的临床菌株blaCTX-M基因，扩增产物经DHPLC分析得到标准菌株和临床菌株色谱峰图，通过比对标准色谱峰图对临床菌株进行基因分型，同时采用单纯随机抽样法选择25株多重PCR扩增阳性菌株进行特异PCR扩增，其产物再进行基因测序来评估DHPLC法的准确性。结果142株产ESBLs的肠杆菌科细菌经多重PCR扩增证实109株携带blaCTX—M基因，检出率为76．8％（109／142）。109株扩增阳性的菌株经DHPLC分析后检出4种不同的blaCTX—M基因型：33株携带CTX-M-3、19株携带CTX-M-15、5株携带CTX—M-9、52株携带CTX—M-14。25株基因测序结果与DHPLC基因分型结果作比较显示：24株DHPLC的基因分型结果与基因测序结果完全吻合，但有1株DHPLC基因分型为CTX-M-15，而测序为CTX—M-82。结论DHPLC可对耐药进行基因快速基因分型，具有准确、快速和经济等优点。Objective To investigate the genotype distribution of CTX-M-type extended-spectrum β-1actamase（ESBLs） by denaturing high-performance liquid chromatography （DHPLC） in Hunan Province and the accuracy of DHPLC assay. Methods The blaCTX-M genes of standard strains and clinical ESBLs- producing Enterobacteriaceae were amplified by multiplex PCR followed by DHPLC and genotype determination. 25 isolates randomly selected were sequenced to assess the accuracy of DHPLC method. Results Among 142 ESBLs-producing isolates, 109 isolates carried blaCTX-M gene （76. 8% ）. Four different CTX-M genotypes were detected by DHPLC, including CTX-M-3 （33 isolates）, CTX-M-15 （19 isolates）, CTX-M-14 （52 isolates） and CTX-M-9 （5 isolates）. The DHPLC typing of 25 isolates suggested that 24 isolates were verified uniformly by the sequencing, but one CTX-M-15 isolate typed by DHPLC was shown to be CTX-M-82 by sequencing. Conclusion DHPLC is a powerful tool for genotyping of the resistance gene and is worth being apphed in the clinical and scientific research with accurate, rapid and economic advantages.

关 键 词：Β内酰胺酶类 大肠杆菌蛋白质类 色谱法 高压液相 基因型 

分 类 号：R686[医药卫生—骨科学]