Purpose To develop a multiplex polymerase chain reaction (PCR) assay to provide a rapid, sensitive and specific method to diagnose staphylococcal infection in cases of suspected septic arthritis.
Methods A novel lysis method was developed to process synovial fluid samples from patients with suspected septic arthritis. Previously designed primers that target three defining 16S rRNA genes in staphylococcus were used for PCR: 16S primers revealed presence of the organism; clfA primers distinguished between S. aureus and coagulase-negative staph; primers in the mecA gene determined the probable susceptibility to methicillin. The PCR was developed to adequate stringency to prevent amplification of any nonstaphylococcal DNA.
Results Based on spiking known quantities of staphylococci into culture-negative synovial fluid, the sensitivity of our method was determined to be 103 CFU/mL. Of 23 clinical samples processed, 2 were positive by PCR for methicillin-sensitive S. aureus, 4 were positive for MRSA, and 17 were negative for staphylococcal DNA. Our results matched those determined by culture-based methods for all samples. Our novel lysis/PCR assay is both efficient and easy to perform, taking only 4.5 hours to complete.
Conclusion This protocol can provide for rapid determination of staphylococci as the etiology of septic arthritis, as well as a determine methicillin sensitivity, making it an excellent candidate for use in the clinical microbiology laboratory.