title: New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci.
abstract: Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was approximately 25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.
authors.affiliation: Centre de Recherche en Infectiologie de l'Université Laval, CHUQ and Division de Microbiologie, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, Canada G1V 4G2.
authors.forename: A
authors.initials: A
authors.lastname: Huletsky
authors.forename: R
authors.initials: R
authors.lastname: Giroux
authors.forename: V
authors.initials: V
authors.lastname: Rossbach
authors.forename: M
authors.initials: M
authors.lastname: Gagnon
authors.forename: M
authors.initials: M
authors.lastname: Vaillancourt
authors.forename: M
authors.initials: M
authors.lastname: Bernier
authors.forename: F
authors.initials: F
authors.lastname: Gagnon
authors.forename: K
authors.initials: K
authors.lastname: Truchon
authors.forename: M
authors.initials: M
authors.lastname: Bastien
authors.forename: F J
authors.initials: FJ
authors.lastname: Picard
authors.forename: A
authors.initials: A
authors.lastname: van Belkum
authors.forename: M
authors.initials: M
authors.lastname: Ouellette
authors.forename: P H
authors.initials: PH
authors.lastname: Roy
authors.forename: M G
authors.initials: MG
authors.lastname: Bergeron
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D016106:Methicillin Resistance / Q000235:genetics*; D008969:Molecular Sequence Data; D009666:Nose / Q000382:microbiology; D046915:Penicillin-Binding Proteins; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; C487789:mecA protein, Staphylococcus aureus