STRATEGY FOR DETECTION AND IDENTIFICATION OF BACTERIA BASED ON 16S RIBOSOMAL-RNA GENES IN SUSPECTED CASES OF WHIPPLES-DISEASE

The 16S ribosomal RNA (rRNA) gene of the phylogenetic subdivision cont aining gram-positive bacteria with a high G+C content was detected spe cifically in clinical specimens from patients suspected of having Whip ple's disease, The primary structure of 16S rDNA amplified from clinic al samples was determined by cloning and sequencing, Two sorts of sequ ences were identified: one corresponded exactly to the rRNA sequence o f Tropheryma, whippelii (GenBank accession no, M87484) while the other was related to that of members of the genus Corynebactium. No sequenc e related to Mycobactevrium spp, or Rhodococcus equi was observed, Exh austive examination of negative specimens with broad-range eubacterial primers detected one sequence related to Enterobacteriaceae and anoth er related to Enterococcus spp, To speed identification of T. whippeli i, a nested amplification method was devised, A first amplification sp ecific for the gram-positive bacteria subdivision was performed, follo wed by a second amplification with T. whippelii-specific primers. The amplified T. whippelii product was checked by digestion with AvaII, St uI, and PstI endonucleases. These techniques were applied to DNA extra cted from seven intestinal biopsy samples, two cerebrospinal fluid sam ples and one articular fluid from patients suspected of having Whipple 's disease, T whippelii 16S rDNA was found in two of the biopsy sample s, one of the cerebrospinal fluid samples and in the articular fluid.