Eight oligonucleotides based upon regions of the small subunit 165 ribosomal RNA gene sequences were analysed against a background of their position within the molecule and their two‐dimensional structure to rationalise their use in recognising Prevotetla intermedia and Prevotetla nigrescens. The 41 clinical isolates from both oral and respiratory sites and two reference strains were subjected to DNA‐DNA hybridisation and multilocus enzyme etectrophoresis to confirm their identity. Alignment of oligonucleotide probes designated I Bi‐2 to I Bi‐6 (for P. intermedia) and 2Bi‐2 (for P. nigrescens) with the 165 rRNA suggested that these probes lacked specificity or were constructed from hypervariable regions. A 52–mer oligonucleotide (designated Bi) retiably detected both species. Because of the high degree of concordance between the I 65 rRNAs of both species, it was necessary to vary the stringency of hybridisation conditions for detection of both species. Thus probe I Bi‐I recognised P. intermedia while I Bi‐l detected both P. intermedia and P. nigrescens at low stringency. However, under conditions of high stringency only P. nigrescens was recognised by probe 2Bi‐I. These probes were highly specific and did not hybridise with DNA from the closety retated P. corporis, nor other periodontal pathogens such as Fuso‐bacterium nucleatum, Actinobacillus actinomycetemcomit‐ans, Treponema denticola and several pigmented species such as Prevotetla metoninogenica, P. denticola, P. loescheii, Porphyramonas osaccharolytica, Py. endodontalis, Py. gin‐givalis, Py. levii, and Py. macacae.
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