Genetic variation in 16S-23S rDNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of Bacteroides species

The structural variation in 16S-23S rDNA internal transcribed spacer region s (ITS) among Bacteroides species was assessed by PCR amplification and seq uencing analysis, and its possible use for molecular diagnosis of these spe cies was evaluated. Ninety strains of the genus Bacteroides, including the species B, distasonis, B, eggerthii, B, fragilis, B, ovatus, B, thetaiotaom icron, B, uniformis and B, vulgatus, produced one to three ITS amplificatio n products with sizes ranging from 615 to 810 bp, Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I, The results of sequence analy sis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-cod ing regions, was highly variable and characteristic for each species, but a common sequence among S, fragilis, B, thetaiotaomicron and B. ovatus was o bserved. A digoxigenin-labeled oligonucleotide probe (named FOT1), which wa s designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B, ovatus . These results suggest that the ITS region is a useful target for the deve lopment of rapid and accurate techniques for identification of Bacteroides species.