Strategy for detection and identification of bacteria based on 16S rRNA genes in suspected cases of Whipple's disease

The 16S ribosomal RNA (rRNA) gene of the phylogenetic subdivision containing gram-positive bacteria with a high G+C content was detected specifically in clinical specimens from patients suspected of having Whipple's disease, The primary structure of 16S rDNA amplified from clinical samples was determined by cloning and sequencing, Two sorts of sequences were identified: one corresponded exactly to the rRNA sequence of Tropheryma, whippelii (GenBank accession no, M87484) while the other was related to that of members of the genus Corynebactium. No sequence related to Mycobactevrium spp, or Rhodococcus equi was observed, Exhaustive examination of negative specimens with broad-range eubacterial primers detected one sequence related to Enterobacteriaceae and another related to Enterococcus spp, To speed identification of T. whippelii, a nested amplification method was devised, A first amplification specific for the gram-positive bacteria subdivision was performed, followed by a second amplification with T. whippelii-specific primers. The amplified T. whippelii product was checked by digestion with AvaII, StuI, and PstI endonucleases. These techniques were applied to DNA extracted from seven intestinal biopsy samples, two cerebrospinal fluid samples and one articular fluid from patients suspected of having Whipple's disease, T whippelii 16S rDNA was found in two of the biopsy samples, one of the cerebrospinal fluid samples and in the articular fluid.