DETECTION OF BACTERIAL DNA BY REAL-TIME PCR USING UNIVERSAL PRIMER AND HYBRIDIZATION PROBE SET: APPLICATIONS FOR INFECTION IN PROSTHETIC JOINT IMPLA NTS

摘要: METHODS: The primers and the hybridization probes were designed with software (LC probe design software, Roche, IN). The target of amplification is the16S rRNA gene, and the primers were designed to detect at least the 7 gram-positive species and 6 gram-negative species that are most frequently associated with prosthesis infection. The hybridization probes were designed to distinguish the gram-positive and gram-negative species by melt analysis. There are several mismatches between the primers/probes and the complementary sequence in the P. acne genome, so designed to avoid detection of this organism. The size of the amplified DNA was 208 bp. Real-time PCR was performed by LightCycler (Roche, IN) for 14 species denoted above, for the extracted DNA samples from 27 surgical specimens of revision cases. All 27 samples were from patients with culture-proven infection after total hip or knee arthroplasty.RESULTS: The limitation of detection was 200 pg of Escherichia coli