title: Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction.
abstract: A multiplex real-time polymerase chain reaction (RT-PCR) targeting the mecA and nuc genes was developed for the detection of methicillin resistance and identification of Staphylococcus aureus. Novel mecA and nuc primers and fluorescence resonance energy transfer hybridization probes specific for the mecA and nuc genes were evaluated. The assay was performed using the LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) and evaluated against the traditional gel-based multiplex PCR (PCR-gel) method currently used at Royal Perth Hospital. Clinical isolates (n = 222) and isolates from a culture collection library (n = 206) were tested by both assays in parallel. The RT-PCR assay was 100% sensitive and specific for the detection of methicillin resistance and for the identification of S. aureus when compared with the PCR-gel assay. Results from the RT-PCR assay showed 5 isolates with lower efficiency fluorescence curves for the nuc gene PCR fragment. DNA sequencing showed mutations within the region of the probe-binding sites compared with the reference strain. The results of the RT-PCR assay were available within 2 h. This rapid mecA/nuc RT-PCR assay is a suitable and practical tool for the routine detection of methicillin resistance and identification of S. aureus, which can be easily incorporated into the diagnostic molecular microbiology laboratory work flow.
authors.affiliation: Department of Microbiology and Infectious Diseases, Royal Perth Hospital, Perth 6847, Western Australia, Australia. anna-marie.costa@health.wa.gov.au
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Costa
authors.forename: Ian
authors.initials: I
authors.lastname: Kay
authors.forename: Silvano
authors.initials: S
authors.lastname: Palladino
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004720:Endonucleases / Q000235:genetics*; D006801:Humans; D016106:Methicillin Resistance; D008836:Micrococcal Nuclease / Q000235:genetics*; D008969:Molecular Sequence Data; D046915:Penicillin-Binding Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; C487789:mecA protein, Staphylococcus aureus; C074002:nuc protein, staphylococcus; D004720:Endonucleases; D008836:Micrococcal Nuclease