运用变性高效液相色谱快速检测TEM型产超广谱β-内酰胺酶菌株  

Rapid detection of TEM-type extended-spectrum β-lactamase by denaturing high performance liquid chromatography

作　　者：刘彩林[1] 孙自镛[1] 汪峰[1] 汪玥[1] 欧国平[1] 廖亚龙[1] 徐令清[1] 孙明月[1] 朱旭慧[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院检验科,武汉430030

出　　处：《国际检验医学杂志》2012年第11期1283-1285,共3页International Journal of Laboratory Medicine

摘　　要：目的建立一种快速检测TEM型产超广谱β-内酰胺酶(ESBLs)临床分离菌及其流行病学监测的新方法。方法采用PCR技术从临床分离株中扩增出TEM型质粒的编码序列,扩增产物用变性高效液相色谱(DHPLC)技术进行分析并进行DNA测序,确定其基因突变的类型,最后通过比对确定其基因型。结果共检测66株临床分离菌,其中42株扩增出TEM型基因。经过DHPLC分析,35株表现为单一的洗脱峰,其形态与TEM-1标准菌株的峰型相一致,测序确定它们的碱基序列亦相一致,不存在变异,为TEM-1型;5株表现为一种异常的洗脱峰,它们均为双峰,形态一致,测序结果表明它们均存在两种相同的基因突变,经比对后确定为TEM-10型;另外2株表现为另一种异常的洗脱峰,测序结果显示为TEM-116型。结论 DHPLC技术可用于细菌耐药基因的分型检测,不但灵敏度和特异度高,而且具有经济、快捷、高通量等特点,具有很大的潜在临床应用价值。Objective To establish a new method to rapidly detect TEM-type ESBLs and epidemiological surveillance.Methods Genotype determination of TEM-type ESBLs was performed by polymerase chain reaction（PCR） amplification from clinical isolates,followed by DHPLC technology and DNA sequencing.Results A total of 66isolates were analyzed,including 42specimens of TEM-type gene were amplified.Through DHPLC,35isolates showed a single elution peak,shape of which was consistent with that of TEM-1standard strain,and sequence of them were also consistent with TEM-1type.5isolates showed an abnormal bimodal elution peaks,and shape and sequencing results showed that there were the same mutations in the NCBI web site,identified as TEM10.2isolates showed another abnormal elution peak and sequencing results same with TEM-116.Conclusion DHPLC technology could be used for typing of bacterial resistance gene detection,might be economic,efficient,high-throughput and with great potential clinical value.

关 键 词：变性高效液相色谱 超广谱Β-内酰胺酶 TEM型 基因分型 

分 类 号：R446.5[医药卫生—诊断学]