Real-time PCR for the rapid detection of vanA and vanB genes.
A real-time PCR assay suitable for use on the Roche LightCycler platform was developed to replace an existing gel-based PCR assay for the simultaneous detection of the vanA & vanB genes in enterococcal isolates. Novel Fluorescence Resonance Energy Transfer (FRET) hybridization probes were designed. The multiplex real-time PCR assay and the existing gel-based assay were 100% concordant and both correctly detected the vanA or vanB genes in 4/4 VanA E. faecium and 25/25 VanB E. faecium. Additionally, 1/1 VanC1 E. gallinarum, 1/1 VanC2 E. casseliflavus and 47/47 vancomycin susceptible enterococci were negative for the vanA and vanB genes in both PCR assays. Results were available within 1.5 h for the real-time PCR assay compared to up to 5.5 h for the conventional PCR assay.
Palladino
Silvano
S
Department of Microbiology & Infectious Diseases, Royal Perth Hospital, Western, Perth, Australia. silvano.palladino@health.wa.gov.au
Kay
Ian D
ID
Costa
Anna Maria
AM
Lambert
Erica J
EJ
Flexman
James P
JP
D001483:Base Sequence; D004352:Drug Resistance, Microbial; D016984:Enterococcus faecium / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005798:Genes, Bacterial / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D010597:Pharmacogenetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D020713:Vancomycin Resistance / Q000235:genetics*