Korean J Lab Med.  2010 Apr;30(2):138-146. 10.3343/kjlm.2010.30.2.138.

Detection of Vancomycin-resistant Enterococci using Multiplex Real-time PCR Assay and Melting Curve Analysis

• Cha CH
• An HK
• Kim JU

• KMID: 1096799
• DOI: http://doi.org/10.3343/kjlm.2010.30.2.138

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.

Keyword

Multiplex real-time PCR; Melting curve analysis; Vancomycin-resistant enterococci

MeSH Terms

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

Figure

• 

  Fig. 1. Melting curve analysis of amplicons obtained from reference strains by using multiplex real-time PCR. Melting curves for Enterococcus faecium (A); Enterococcus faecalis (B); E. faecium/vanA (C); E. faecalis/vanB (D); Enterococcus gallinarum/vanC1 (E); and Enterococcus casseliflavus/vanC2/C3 (F) are shown.

• 

  Fig. 2. Melting curve analysis for the identification of mixed strains. (A) Melting curve for DNA extracted from black colonies growing on the Enterococcal agar plate. VanA, ddl Enterococcus faecalis, and ddl Enterococcus faecium peaks are shown. (B) Melting curve for DNA extracted from pure colonies of E. faecium with phenotype VanA growing on the blood agar plate. Peaks for E. faecium with VanA and ddl genes. (C) Melting curve for DNA extracted from pure colonies of E. faecalis with phenotype VanA growing on the blood agar plate. Peaks for E. faecalis with VanA and ddl genes.

• 

  Fig. 3. Melting curve analysis of amplicons obtained from 3 mixed strains of 2 or more enterococci by using multiplex real-time PCR. Melting curves for Enterococcus faecalis and Enterococcus faecium with vanA gene (A); E. faecalis and E. faecium with vanA gene and Enterococcus gallinarum with vanC1 gene (B); and Enterococcus casseliflavus/Enterococcus flavescens with vanC2/C3 gene and E. gallinarum with vanC1 gene (C).

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