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Bacteriology

Rapid and Accurate Identification of Coagulase-Negative Staphylococci by Real-Time PCR

K. J. Edwards, M. E. Kaufmann, N. A. Saunders
K. J. Edwards
Molecular Biology Unit, Virus Reference Division, and
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M. E. Kaufmann
Laboratory of Hospital Infection, Central Public Health Laboratory, Colindale, London, NW9 5HT, United Kingdom
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N. A. Saunders
Molecular Biology Unit, Virus Reference Division, and
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DOI: 10.1128/JCM.39.9.3047-3051.2001
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    Fig. 1.

    Biprobe peak profiles. (A) Peak profiles obtained with biprobe A. Peak 0 melted at 58°C, peak 1 melted at 52°C, and peak 2 melted at 50°C. (B) Peak profiles obtained with biprobe B. Peak 0 melted at 64°C, peak 1 melted at 60°C, peak 2 melted at 56°C, peak 3 melted at 58°C, peak 4 melted at 53°C, and peak 5 melted at 52°C. (C) Peak profiles obtained with biprobe C. Peak 0 melted at 68°C, peak 1 melted at 60°C, peak 2 melted at 56°C, and peak 3 melted at 54°C. (D) Peak profiles obtained with biprobe D. Peak 0 melted at 60°C, and peak 1 melted at 58°C.

Tables

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  • Table 1.

    Type strains used in the study

    SpeciesStrain
    S. arlettaeNCTCa 12413
    S. aureusNCTC 8523
    S. auricularisNCTC 12101
    S. capitisNCTC 11045
    S. capraeNCTC 12196
    S. chromogenesNCTC 10530
    S. cohniiNCTC 11041
    S. delphiniNCTC 12225
    S. epidermidisNCTC 11047
    S. equorumNCTC 12414
    S. gallinarumNCTC 12195
    S. haemolyticusNCTC 11042
    S. hominisNCTC 11320
    S. hyicusNCTC 10350
    S. intermediusNCTC 11048
    S. kloosiiNCTC 12415
    S. lentusNCTC 12102
    S. lugdunensisNCTC 12217
    S. saprophyticusNCTC 7292
    S. sciuriNCTC 12103
    S. schleiferiNCTC 12218
    S. simulansNCTC 11046
    S. warneriNCTC 11044
    S. xylosusNCTC 11043
    Stomatococcus mucilaginosusNCTC 10663
    Micrococcus luteusNCTC 2665
    • ↵a NCTC, National Collection of Type Cultures (Central Public Health Laboratory, London, England).

  • Table 2.

    DNA sequences of primers and biprobes

    Name of primer or probeSequence
    Primers
     STAR15′-GCG GdT CCA TCT ATA AGT GA-3′a
     STAF15′-GGG TGA GTA ACA CGT GGA-3′
     STAF25′-GGG TGA GTA ACA CGT GGG-3′
     STBF15′-ATT CGA AGC AAC GCG AAG-3′
     STBR15′-CCA TGC ACC ACC TGT CAC-3′
    Probes
     CNS Probe A5′-GGA TAA TAT ATT GAA CCG CA-3′
     CNS Probe B5′-TCC GGT TTC CCG AAG TTG TC-3′
     CNS Probe C5′-GAG CGG TCA AAG GAT GTC AAG-3′
     CNS Probe D5′-CCT CTG ATC CCT CTA GAA ATA-3′
    • ↵a d, A or G.

  • Table 3.

    Biprobe identification scheme for CNSa

    OrganismBiprobe melting peakb obtained with:
    CNS Probe ACNS Probe BCNS Probe CCNS Probe D
    S. epidermidis0331
    S. warneri110–
    S. lugdunensis200–
    S. aureus232–
    S. intermedius240–
    S. chromogenes243–
    S. sciuri250–
    S. hominis–1 or 21–
    S. haemolyticus–22–
    S. capitis–330 or 1
    S. caprae–331
    S. schleiferi–40–
    S. simulans–42–
    S. saprophyticus–43–
    S. cohnii–52–
    • ↵a Results highlighted in bold are not necessary for identification of that species. –, no peak observed.

    • ↵b Designated 0 through 5, as defined in the legend to Fig. 1.

  • Table 4.

    Results of testing of 142 clinical isolates by ID 32 Staph and PCR and by real-time PCR

    OrganismTotal no. of isolatesNo. of isolates identified by:
    ID 32 Staph and PCRReal-time PCR16S rRNA sequencing
    S. hominis171717–b
    S. sciuri333–
    S. cohnii222–
    S. intermedius222–
    S. simulans555–
    S. epidermidis3026304
    S. aureus1110111
    S. saprophyticus6561
    S. warneri111311–
    S. haemolyticus242724–
    S. schleiferi464–
    S. lugdunensis101210–
    S. capitis121471
    S. arlettae1001
    S. pasteuri1001
    Staphylococcussp.1001
    Acinetobacter lwoffii1001
    Micrococcus luteus1001
    • a Number of isolates identified using a combination of all tests and using 16S rRNA sequencing to confirm identity when discrepancies occurred between the ID 32 Staph and PCR and the real-time PCR identifications.

    • ↵b –, not performed.

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Rapid and Accurate Identification of Coagulase-Negative Staphylococci by Real-Time PCR
K. J. Edwards, M. E. Kaufmann, N. A. Saunders
Journal of Clinical Microbiology Sep 2001, 39 (9) 3047-3051; DOI: 10.1128/JCM.39.9.3047-3051.2001

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Rapid and Accurate Identification of Coagulase-Negative Staphylococci by Real-Time PCR
K. J. Edwards, M. E. Kaufmann, N. A. Saunders
Journal of Clinical Microbiology Sep 2001, 39 (9) 3047-3051; DOI: 10.1128/JCM.39.9.3047-3051.2001
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KEYWORDS

Coagulase
polymerase chain reaction
Staphylococcal Infections
Staphylococcus

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