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We have developed an assay based on a 16S rDNA broad-range amplification system followed by species-specific detection with a commercially available PCR-ELISA kit. B. burgdorferi and S. pneumoniae were used as model systems for arthritis and meningitis, respectively. The sensitivity of the B. burgdorferi assay was comparable to that of a species-specific PCR, whereas for S. pneumoniae the detection limit was one to three organisms as determined by plate counts. To specifically differentiate two species, two discontinuously located nucleotide differences in the region complementary to the capture probe are required during the detection step with the PCR-ELISA kit. A preliminary clinical evaluation was performed with eight specimens (joint and cerebrospinal fluids) previously shown to contain B. burgdorferi DNA. Except for one sample which was positive by the broad-range PCR-ELISA system only, the results were in agreement with those obtained by B. burgdorferi species-specific PCR. None of the 23 control samples were positive by either method. Thus, broad-range amplification in combination with the PCR-ELISA kit promises to be a sensitive and specific format for the detection of agents causing reactive arthritis, meningitis or other diseases associated with a limited number of different bacteria.