DETECTION AND IDENTIFICATION OF BACTERIA USING IN-HOUSE BROAD RANGE 16S RDNA PCR AMPLIFICATION AND GENUS-SPECIFIC DNA HYBRIDIZATION PROBES,LOCATED WITHIN VARIABLE REGIONS OF 16S RIBOSOMAL-RNA GENES

Broad range PCR amplification and genus-specific 16S ribosomal DNA hyb ridization was used to demonstrate that Chlamydia, Helicobacter and Mo biluncus hybridization probes, located within variable regions V3, V4, and V9 of the 16S rDNA, specifically bound to the corresponding PCR p roduct obtained from pure cultures of the three genera. The sensitivit y of the assay was determined by analysis of C. trachomatis serially d iluted in urine. The detection limit was 1-10 elementary bodies using a hybridization probe derived from the variable region V3 of the 16S r RNA gene. A PCR product was furthermore formed in urine specimens not containing C. trachomatis, showing amplification of chlamydia also in the presence of DNA from the resident urethral flora that competes for annealing sites. Analysis of a restricted number of male urine specim ens using the C. trachomatis-specific probe showed complete agreement with culture and a commercially available PCR kit. Our method not only has the capacity to detect C. trachomatis in microbiologically mixed urine samples but also the potential advantage of identifying other ba cterial pathogens from the same PCR product by varying the hybridizati on probes.