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

Abstract

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. Key words: Enterococcus; Vancomycin resistance; Genes; Genotype