[Detection of vancomycin-resistant enterococci using multiplex real-time PCR assay and melting curve analysis].
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.
Cha
Choong Hwan
CH
Department of Laboratory Medicine, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Korea.
An
Hae Kyong
HK
Kim
Jeong Uk
JU
D001426:Bacterial Proteins / Q000235:genetics; D019729:Carbon-Oxygen Ligases / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D016983:Enterococcus / Q000235:genetics / Q000302:isolation & purification*; D013293:Enterococcus faecalis / Q000235:genetics / Q000302:isolation & purification; D016984:Enterococcus faecium / Q000235:genetics / Q000302:isolation & purification; D005838:Genotype; D009691:Nucleic Acid Denaturation; D010453:Peptide Synthases / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D020713:Vancomycin Resistance / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C071437:VanA ligase, Bacteria; C079913:VanB protein, Enterococcus; D019729:Carbon-Oxygen Ligases; D010453:Peptide Synthases; C108395:VanC protein, Enterococcus; C019638:D-alanylalanine synthetase