肠球菌万古霉素高水平耐药基因vanA、vanB、vanD和vanM快速分型检测  

Rapid detection of high-level vancomycin-resistant genes, vanA, vanB, vanD and vanM, in enterococci

作　　者：林东昉[1] 陈春辉[1] 周迎[1] 徐晓刚[1] 

机构地区：[1]复旦大学附属华山医院抗生素研究所,上海200040

出　　处：《中华传染病杂志》2017年第2期99-104,共6页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金（81171613）

摘　　要：目的 建立基于多重PCR技术的肠球菌万古霉素高水平耐药基因vanA、vanB、vanD和vanM的快速分型检测方法。方法 通过分析可介导肠球菌万古霉素高度耐药的D-丙氨酸∶D-乳酸（D-Ala∶D-Lac）连接酶基因序列差异，设计可同时检测肠球菌万古霉素高水平耐药基因vanA、vanB、vanD和vanM的多重PCR分型检测方法，以含vanA、vanB、vanD和vanM基因的重组质粒为阳性对照，以临床常见病原菌DNA为阴性对照，评价其敏感度及特异度。采用新建方法检测50株万古霉素耐药临床分离株，50株万古霉素耐药肠球菌（VRE）临床株于2006年1月至2014年12月分离自上海地区9家医院，并与常规PCR及测序方法比较。结果 vanA、vanB、vanD和vanM基因核苷酸序列一致率为60.8%～71.3%，根据序列差异建立的分型方法可对不同基因型阳性对照样品进行准确分型。所有阴性对照样品均未出现假阳性。检测50株临床分离VRE，18株为vanA型，32株为vanM型，与常规PCR及测序方法比较，敏感度及特异性度均为100.0%。检测下限2×10拷贝/PCR反应，检测可在3.5 h内完成。结论 建立了一种基于多重PCR技术的VRE万古霉素高水平耐药基因快速分型方法，可用于VRE菌株的分子流行病学研究及检测。Objective To develop a multiple polymerase chain reaction （PCR） technique based assay for rapid detection of vanA, vanB, vanD and vanM in high-level vancomycin-resistant enterococci.Methods After analyzing the uncleotide sequence divergence among D-Ala∶D-Lac ligase genes, an multiplex PCR assay for vanA, vanB, vanD and vanM genes in high-level vancomycin-resistant enterococci were designed. By using recombination plasmids containing vanA, vanB, vanD and vanM genes as positive control, and non-vancomycin resistant enterococci （non-VRE） common pathogenic bacterial DNA as negative control, the sensitivity and specificity of the assay were evaluated. Fifty vancomycin-resistant enterococci （VRE） isolates were detected by the assay. Fifty clinical strains of VRE were isolated from 9 hospitals in Shanghai from January 2006 to December 2014. The results were compared with the conventional PCR and sequencing methods.Results The identity of the D-Ala∶D-Lac ligase genes were 60.8%—71.3% of vanA, vanB, vanD and vanM genes. The multiplex PCR assay could identify the genotypes of the positive control samples accurately. No false positive results were found in negative control samples. Among fifty VRE strains detected by the assay, 18 were vanA genotype and 32 were vanM genotype. Comparison of the multiplex PCR assay and sequencing methods revealed sensitivity and specificity of 100%. The detection limit of the assay was 2×10 copies/PCR reaction. The experiment could be done within 3.5 h.Conclusions A multiplex PCR assay is developed to rapid identify the genotype of the high-level vancomycin-resistant enterococci, which can be used for the molecular epidemiology research and detection of VRE.

关 键 词：肠球菌属 万古霉素抗药性 基因 基因型 

分 类 号：R440[医药卫生—诊断学]