[Detection of methicillin-resistant Staphylococcus aureus using PCR and non-radioactive DNA probe]

Two commonly used diagnostic tests for Staphylococcus aureus (MRSA) are cultivation of bacteria from clinical samples on mannitol-salt agar plate or on MRSA-screen agar plate with oxacillin. However, the use of PCR and DNA probes is considered more rapid and sensitive, as staphylococcal cells have high resistance to beta-lactam due to the novel penicillin-binding protein, PBP2'. Therefore, three different pairs of DNA primers (P1F-P1R, P2F-P2R and P3F-P3R) complementary with unique regions of the MRSA PBP2' gene were synthesized for use in polymerase chain reactions with DNA of MRSA. Amplified target DNA of 466, 480 and 630 bp could be resolved on ethidium bromide-stained gels, and hybridized with DNA probes conjugated to alkaline phosphatase. When applied to pure cultures on the MRSA screen agar, the DNA probes detected MRSA in 180 of 196 (P1F-P1R), 72 of 83 (P2F-P2R) and 66 of 71 (P3F-P3R) culture-positive specimens (accuracy range, 86.7-93.0%), while 61.6-89.3% of MRSA were detectable in culture-positive specimens streaked on the mannitol-salt agar. When applied directly to clinical samples, this DNA probe assay detected MRSA in culture-positive specimens within an accuracy range of 50.0-79.3%. It was thus clear that comparison of cultivation and DNA hybridization results yields good correlation with respect to detection of MRSA. These results suggest that the DNA probe assay may be useful in complementing existing culture techniques.