Detection of Vancomycin-resistant Enterococci using Multiplex Real-time PCR Assay and Melting Curve Analysis.
10.3343/kjlm.2010.30.2.138
- Author:
Choong Hwan CHA
1
;
Hae Kyong AN
;
Jeong Uk KIM
Author Information
1. Department of Laboratory Medicine, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Korea. jukim@gnah.co.kr
- Publication Type:Original Article ; English Abstract ; Evaluation Studies
- Keywords:
Multiplex real-time PCR;
Melting curve analysis;
Vancomycin-resistant enterococci
- MeSH:
Bacterial Proteins/genetics;
Carbon-Oxygen Ligases/genetics;
DNA, Bacterial/genetics;
Enterococcus/genetics/*isolation &purification;
Enterococcus faecalis/genetics/isolation &purification;
Enterococcus faecium/genetics/isolation &purification;
Genotype;
Nucleic Acid Denaturation;
Peptide Synthases/genetics;
Phenotype;
*Polymerase Chain Reaction;
Vancomycin Resistance/*genetics
- From:The Korean Journal of Laboratory Medicine
2010;30(2):138-146
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
BACKGROUND: We developed and evaluated the utility of a multiplex real-time PCR assay that uses melting curve analysis and allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. METHODS: The specificity of the assay was tested using 4 reference strains of vancomycin-resistant enterococci (VRE) and 2 reference strains of vancomycin-susceptible enterococci. Ninety-three clinical isolates of enterococci with different glycopeptide-resistant phenotypes were genotyped and identified using a multiplex real-time PCR assay and melting curve analysis. RESULTS: Representative melting curves were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis, Enterococcus gallinarum, and Enterococcus casseliflavus. Phenotypic and genotypic analysis of the isolates revealed same results for 82 enterococcal isolates, while in 4 isolates, the glycopeptide-resistant phenotypes were inconsistent with the glycopeptide-resistant genotypes and in the 4 other isolates, species could not be accurately identified. Three isolates with mixed strains, which were detected by the PCR assay, could not be correctly identified using phenotypic methods. CONCLUSIONS: VRE genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using multiplex real-time PCR assay and melting curve analysis.
