PRELIMINARY-STUDY USING SPECIES-SPECIFIC OLIGONUCLEOTIDE PROBE FOR RIBOSOMAL-RNA OF BILOPHILA-WADSWORTHIA

Portions of the 16S RNA from a urease-positive Bilophila wadsworthia s train were sequenced, and a probe was constructed. The probe was end l abeled with [P-32]ATP and polynucleotide kinase and hybridized on a ny lon filter (by dot blot hybridization) to the immobilized rRNA of 12 B . wadsworthia strains and eight other anaerobic isolates. The probe ef ficiently?v hybridized only to the Bilophila strains. Cross-reactivity at high RNA levels (2,000 ng) was observed with one strain of Bactero ides thetaiotaomicron and one strain of Bacteroides fragilis (with 10X SET buffer [20x SET buffer is 0.5 M NaCl, 0.03 M Tris, and 2 mM EDTA] ) but was not seen at lower RNA levels or with 5x SET buffer. When tes ted against mixed cultures of aerobic and anaerobic isolates represent ative of appendiceal abscess flora, the probe did not react with mixed cultures containing no Bilophila cells and could detect greater than or equal to 10(5) Bilophila CFU/ml when the mixture was seeded with Bi lophila cells. This probe is of potential use in the rapid identificat ion of pure isolates and in the direct identification of B. wadsworthi a in clinical specimens.