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Journal of Clinical Microbiology, November 2009, p. 3692-3706, Vol. 47, No. 11
0095-1137/09/$08.00+0     doi:10.1128/JCM.00766-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Multiplex Real-Time PCR for Rapid Staphylococcal Cassette Chromosome mec Typing {triangledown}

Liang Chen,1 José R. Mediavilla,1 Duarte C. Oliveira,2 Barbara M. Willey,3 Herminia de Lencastre,4,5 and Barry N. Kreiswirth1*

Public Health Research Institute Center, International Center for Public Health, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103,1 CREM—Center for Microbiological Resources, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica,2 Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Oeiras, Portugal,5 Department of Microbiology, Mount Sinai Hospital, Toronto, Ontario, Canada,3 Laboratory of Microbiology, The Rockefeller University, New York, New York 100654

Received 15 April 2009/ Returned for modification 16 June 2009/ Accepted 26 August 2009

Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets (mec class, ccr) for each SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the {Delta}mecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the {Delta}mecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital- and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCCmec typing of MRSA isolates.

* Corresponding author. Mailing address: PHRI TB Center, International Center for Public Health, 225 Warren Street, Newark, NJ 07103. Phone: (973) 854-3240. Fax: (973) 854-3101. E-mail: kreiswba{at}umdnj.edu

{triangledown} Published ahead of print on 2 September 2009.

Journal of Clinical Microbiology, November 2009, p. 3692-3706, Vol. 47, No. 11
0095-1137/09/$08.00+0     doi:10.1128/JCM.00766-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Varshney, A. K., Mediavilla, J. R., Robiou, N., Guh, A., Wang, X., Gialanella, P., Levi, M. H., Kreiswirth, B. N., Fries, B. C. (2009). Diverse Enterotoxin Gene Profiles among Clonal Complexes of Staphylococcus aureus Isolates from the Bronx, New York. Appl. Environ. Microbiol. 75: 6839-6849 [Abstract] [Full Text]  


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