"Reverse" DNA hybridization method for the rapid identification of subgingival microorganisms.

Abstract

A "reverse" hybridization method is described, in which whole chromosomal DNA was extracted from 10-20 colonies of "unknown" strains in pure culture and labelled with digoxigenin by a random primer technique. DNA probes were prepared from a total of 23 strains and hybridized with targets containing 100 ng purified, denatured DNA from 38 reference strains fixed to nitrocellulose. 21/23 digoxigenin-labelled DNA probes successfully detected all members of the homologous species present on filters. Probes to Fusobacterium nucleatum strains 364 and MG detected 3/4 and 1/4 members of this species, respectively; 13/23 probes were 100% specific, but cross reactions between 10 probes and DNA targets from closely related, heterologous species occurred in 15/834 possible instances. False-positive reactions that occurred between closely related species were, however, easily distinguished and did not prevent the accurate identification of probe strains. Digoxigenin-labelled probes were capable of detecting 100 pg of homologous DNA. The reverse hybridization procedure allows identification or grouping of a large number of isolates within 3 days and provides a more economical means of characterizing subgingival isolates than predominant cultivable techniques and conventional phenotypic testing. This method could be adapted for the direct identification of microorganisms in subgingival plaque samples.