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Journal of Clinical Microbiology, January 2007, p. 141-146, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01228-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid Multiplex PCR Assay for Identification of USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates{triangledown}

Kristin K. Bonnstetter,1 Daniel J. Wolter,1 Fred C. Tenover,2 Linda K. McDougal,2 and Richard V. Goering1*

Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska 68178,1 Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Atlanta, Georgia 303332

Received 15 June 2006/ Returned for modification 5 August 2006/ Accepted 27 October 2006

Recent reports have noted a discernible increase in the number of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections in patients without traditional risk factors. In the United States, the most prominent CA-MRSA strain encodes Panton-Valentine leukocidin (PVL) cytotoxin genes, belongs to pulsed field gel electrophoresis type USA300 and multilocus sequence type 8, and carries staphylococcal cassette chromosome mec (SCCmec) type IV. At present, molecular characterization of MRSA strains, such as USA300, can be time-consuming and is often beyond the technical capability of many clinical laboratories, making routine identification difficult. We analyzed the chromosomal regions flanking the SCCmec element in 44 USA300 MRSA isolates and identified a signature "AT repeat" sequence within the conserved hypothetical gene SACOL0058 located 1.4 kb downstream of the 3' end of the J1-SCCmec chromosomal junction. Only USA300 isolates tested contained a sequence of ≥6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had ≥6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for ≥6 AT repeats in combination with primers to detect PVL, we developed a multiplex PCR assay specific for the identification of USA300 strains. Multiplex results were 100% concordant with DNA sequencing, suggesting that the method has promise as a means of rapidly identifying USA300 isolates.

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178. Phone: (402) 280-4098. Fax: (402) 280-1875. E-mail: rgoering{at}creighton.edu.

{triangledown} Published ahead of print on 8 November 2006.

Journal of Clinical Microbiology, January 2007, p. 141-146, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01228-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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