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Journal of Clinical Microbiology, September 2001, p. 3332-3338, Vol. 39, No. 9
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.9.3332-3338.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Multiplex PCR Protocol for the Diagnosis of Staphylococcal Infection

William J. Mason,1 Jon S. Blevins,1 Karen Beenken,1 Noroyono Wibowo,2 Neelum Ojha,3 and Mark S. Smeltzer1,*

Department of Microbiology and Immunology1 and Clinical Microbiology Laboratory,3 University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, and Department of Obstetrics and Gynecology, University of Indonesia, Jakarta, Indonesia2

Received 27 December 2000/Returned for modification 16 February 2001/Accepted 6 July 2001

We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection. The protocol was designed to (i) detect any staphylococcal species to the exclusion of other bacterial pathogens (based on primers corresponding to Staphylococcus-specific regions of the 16S rRNA genes), (ii) distinguish between S. aureus and the coagulase-negative staphylococci (CNS) (based on amplification of the S. aureus-specific clfA gene), and (iii) provide an indication of the likelihood that the staphylococci present in the specimen are resistant to oxacillin (based on amplification of the mecA gene). The expected fragments were amplified from each of 60 staphylococcal isolates (13 oxacillin-resistant S. aureus isolates, 23 oxacillin-sensitive S. aureus isolates, 17 oxacillin-resistant CNS, and 7 oxacillin-sensitive CNS). No amplification products were observed with template DNA from nonstaphylococcal species, and the efficiency of amplification of staphylococcal targets was not adversely affected by the presence of DNA from other bacterial species in the same sample. The utility of the protocol for the analysis of clinical samples was verified by analysis of aliquots taken directly from BacT/Alert blood culture bottles. Of 77 blood cultures tested, only 7 yielded results inconsistent with those of conventional methods of diagnosis and susceptibility testing. Of those, one was identified as a CNS species by PCR and S. aureus by conventional methods. We also identified two isolates that were mecA positive but were oxacillin sensitive according to conventional methods. The other four samples failed to yield any amplification product even with a control set of primers corresponding to a conserved region of the eubacterial rRNA genes.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Mail Slot 511, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205. Phone: (501) 686-7958. Fax: (501) 686-5359. E-mail: smeltzermarks{at}uams.edu.

Journal of Clinical Microbiology, September 2001, p. 3332-3338, Vol. 39, No. 9
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.9.3332-3338.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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