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Journal of Clinical Microbiology, November 2004, p. 4947-4955, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.4947-4955.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

New Quadriplex PCR Assay for Detection of Methicillin and Mupirocin Resistance and Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci

Kunyan Zhang,1,2,3,4,5* Jennifer Sparling,1 Barbara L. Chow,1,5 Sameer Elsayed,2,3,5 Zafar Hussain,6 Deirdre L. Church,2,4,5 Daniel B. Gregson,2,4,5 Thomas Louie,3,4 and John M. Conly1,2,3,4,5

Centre for Antimicrobial Resistance, Calgary Health Region—Calgary Laboratory Services—University of Calgary,1 Departments of Pathology and Laboratory Medicine,2 Microbiology and Infectious Diseases,3 Medicine,4 University of Calgary, and Division of Microbiology, Calgary Laboratory Services, Calgary, Alberta,5 Department of Microbiology and Infection Control, London Health Science Centre, London, Ontario, Canada6

Received 26 March 2004/ Returned for modification 8 June 2004/ Accepted 22 July 2004

Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin- and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin- and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin- and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.

* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, University of Calgary, 9-3535 Research Rd. N.W., Calgary, Alberta, Canada T2L 2K8. Phone: (403) 770-3583. Fax: (403) 770-3347. E-mail: kunyan.zhang{at}cls.ab.ca.

Journal of Clinical Microbiology, November 2004, p. 4947-4955, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.4947-4955.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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