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Journal of Clinical Microbiology, May 2004, p. 1875-1884, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1875-1884.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

New Real-Time PCR Assay for Rapid Detection of Methicillin- Resistant Staphylococcus aureus Directly from Specimens Containing a Mixture of Staphylococci

A. Huletsky,1,2 R. Giroux,1 V. Rossbach,1 M. Gagnon,1 M. Vaillancourt,1 M. Bernier,1 F. Gagnon,1 K. Truchon,3 M. Bastien,1 F. J. Picard,1 A. van Belkum,4 M. Ouellette,1,2 P. H. Roy,1,5 and M. G. Bergeron1,2*

Centre de Recherche en Infectiologie de l'Université Laval, CHUQ,1 Division de Microbiologie, Faculté de Médecine,2 Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Sainte-Foy, Québec, Canada G1V 4G2,5 Infectio Diagnostic (I.D.I.) Inc., Sainte-Foy, Québec, Canada G1V 2K8,3 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, 3015 GD Rotterdam, The Netherlands4

Received 16 July 2003/ Returned for modification 8 October 2003/ Accepted 10 February 2004

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 ~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.

* Corresponding author. Mailing address: Centre de Recherche en Infectiologie de l'Université Laval, CHUQ (Pavillon CHUL), 2705 boul. Laurier, Sainte-Foy, Québec G1V 4G2, Canada. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: michel.g.bergeron{at}crchul.ulaval.ca.

Journal of Clinical Microbiology, May 2004, p. 1875-1884, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1875-1884.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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