title: Species-level identification of staphylococcal isolates by real-time PCR and melt curve analysis.
abstract: A real-time PCR assay was developed to identify common staphylococcal species. A single set of consensus primers was designed to amplify a portion of the 16S rRNA gene, and a pair of fluorescence resonance energy transfer probes was used to identify species based on the unique melt properties of the probes resulting from sequence variations in the amplicons from each species. Nine common staphylococcal strains (S. aureus, S. capitis, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, S. schleiferi, S. simulans, and S. warneri) were used for assay development. The species-specific melting profiles were validated by correctly identifying 36 of 37 coagulase-negative staphylococcal (CoNS) isolates identified by ribotyping. In a study of clinical isolates, the PCR/melt curve approach correctly identified 56/56 S. aureus isolates identified by coagulase/protein A latex agglutination. Fifty-four CoNS clinical isolates characterized using the API Staph assay were studied, with the PCR/melt curve approach yielding matching identifications for 32/54 (59%). The API Staph assay was unable to identify 18 CoNS isolates, and differing results were obtained for 4 isolates. Sequencing of the 22 discrepant or unidentified CoNS samples revealed that the PCR/melt curve results were correct for all but one isolate. Thus, PCR/melt curve analysis achieved a nearly 100% accuracy and performed better than biochemical testing. Performance of the PCR/melt curve approach requires less than 2 h after colony selection. This method thus provides a rapid and accurate approach to the identification of staphylococcal species in the clinical laboratory.
authors.affiliation: Department of Pathology, Evanston Northwestern Healthcare and Northwestern University Feinberg School of Medicine, 2650 Ridge Avenue, Evanston, IL 60201, USA.
authors.forename: Aine
authors.initials: A
authors.lastname: Skow
authors.forename: Kathy A
authors.initials: KA
authors.lastname: Mangold
authors.forename: Mohammed
authors.initials: M
authors.lastname: Tajuddin
authors.forename: Anne
authors.initials: A
authors.lastname: Huntington
authors.forename: Brett
authors.initials: B
authors.lastname: Fritz
authors.forename: Richard B
authors.initials: RB
authors.lastname: Thomson
authors.forename: Karen L
authors.initials: KL
authors.lastname: Kaul
mesh_terms: D015373:Bacterial Typing Techniques; D001769:Blood / Q000382:microbiology; D015342:DNA Probes; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013045:Species Specificity; D013203:Staphylococcal Infections / Q000382:microbiology*; D013210:Staphylococcus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D044366:Transition Temperature
chemical_list: D015342:DNA Probes; D012336:RNA, Ribosomal, 16S