Multiplex PCR protocol for the diagnosis of staphylococcal infection

We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection. The protocol was designed to (i) detect any staph ylococcal species to the exclusion of other bacterial pathogens (based on p rimers corresponding to Staphylococcus-specific regions of the 16S rRNA gen es), (ii) distinguish between S. aureus and the coagulase-negative staphylo cocci (CNS) (based on amplification of the S. aureus-specific clfA gene), a nd (Iii) provide an indication of the likelihood that the staphylococci pre sent in the specimen are resistant to oxacillin (based on amplification of the mecA gene). The expected fragments were amplified from each of 60 staph ylococcal 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 f rom nonstaphylococcal species, and the efficiency of amplification of staph ylococcal targets was not adversely affected by the presence of DNA from ot her bacterial species in the same sample. The utility of the protocol for t he 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 CN S 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 amplif ication product even with a control set of primers corresponding to a conse rved region of the eubacterial rRNA genes.