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In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiateYersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three differentYersinia species and partial 23S rDNA sequences for threeY. pestis and threeY. pseudotuberculosis strains were determined. While 16S rDNA sequences ofY. pestis andY. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection ofYersinia species by PCR and in situ hybridization, respectively. As few as 102 Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation ofY. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenicYersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenicYersinia species (in particularY. pestis andY. pseudotuberculosis ) was developed. The applicability of this technique was demonstrated by detection ofY. pestis andY. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifyingY. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.

Development of rRNA-Targeted PCR and In Situ Hybridization with Fluorescently Labelled Oligonucleotides for Detection of Yersinia Species