title: Development of a real-time PCR assay for rapid identification of methicillin-resistant Staphylococcus aureus from clinical samples.
abstract: A major drawback of mecA PCR to detect methicillin-resistant Staphylococcus aureus (MRSA) directly from patient materials is the high frequency of methicillin-resistant coagulase-negative staphylococci. Therefore, a reliable detection method for MRSA from clinical samples using real-time PCR was developed. The PCR assay targeting the integration site (orfX) of the staphylococcal cassette chromosome mec (SCCmec) was evaluated in MRSA SCCmec reference strains (n = 9), MRSA ST strains (n = 16) and clinical isolates of MRSA (n = 124), MSSA (n = 53), methicillin-resistant coagulase-negative staphylococci (n = 47), and methicillin-susceptible, coagulase-negative staphylococci (n = 32). The diagnostic values of the assay were 98% sensitivity and 100% specificity. Furthermore, the PCR detection method was evaluated with 60 swabs from different body sites which were incubated overnight in brain-heart infusion. The PCR gave positive results for 27 of 29 swabs which were found to contain MRSA by conventional methods. The diagnostic values of the PCR assay for these samples were 93% sensitivity and 100% specificity. To determine the in vitro sensitivity of the assay, swabs were inoculated with serially diluted bacterial suspensions. After overnight enrichment the detection limit of the PCR was less than 10 CFU/swab. This new real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine microbiological laboratory.
authors.affiliation: Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Trogerstr. 9, D-81675 Munich, Germany.
authors.forename: Ralf M
authors.initials: RM
authors.lastname: Hagen
authors.forename: Irene
authors.initials: I
authors.lastname: Seegmüller
authors.forename: Jila
authors.initials: J
authors.lastname: Navai
authors.forename: Ines
authors.initials: I
authors.lastname: Kappstein
authors.forename: Norbert
authors.initials: N
authors.lastname: Lehn
authors.forename: Thomas
authors.initials: T
authors.lastname: Miethke
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000032:analysis; D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D005798:Genes, Bacterial; D006801:Humans; D008712:Methicillin / Q000494:pharmacology; D016106:Methicillin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013203:Staphylococcal Infections / Q000175:diagnosis*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D014157:Transcription Factors / Q000032:analysis
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D017931:DNA Primers; D015342:DNA Probes; C414317:SarH1 protein, Staphylococcus aureus; D014157:Transcription Factors; D008712:Methicillin