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Journal of Clinical Microbiology
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Bacteriology

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

Karlheinz Trebesius, Dag Harmsen, Alexander Rakin, Jochen Schmelz, Jürgen Heesemann
Karlheinz Trebesius
Max-von-Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, D-80336 Munich, and
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Dag Harmsen
Institut für Hygiene und Mikrobiologie, Universität Würzburg, D-97080 Würzburg, Germany
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Alexander Rakin
Max-von-Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, D-80336 Munich, and
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Jochen Schmelz
Institut für Hygiene und Mikrobiologie, Universität Würzburg, D-97080 Würzburg, Germany
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Jürgen Heesemann
Max-von-Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, D-80336 Munich, and
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DOI: 10.1128/JCM.36.9.2557-2564.1998
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ABSTRACT

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. 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 102Yersinia 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 of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenicYersinia species (in particular Y. pestis andY. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestisand Y. 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.

  • Copyright © 1998 American Society for Microbiology
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Development of rRNA-Targeted PCR and In Situ Hybridization with Fluorescently Labelled Oligonucleotides for Detection of Yersinia Species
Karlheinz Trebesius, Dag Harmsen, Alexander Rakin, Jochen Schmelz, Jürgen Heesemann
Journal of Clinical Microbiology Sep 1998, 36 (9) 2557-2564; DOI: 10.1128/JCM.36.9.2557-2564.1998

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Development of rRNA-Targeted PCR and In Situ Hybridization with Fluorescently Labelled Oligonucleotides for Detection of Yersinia Species
Karlheinz Trebesius, Dag Harmsen, Alexander Rakin, Jochen Schmelz, Jürgen Heesemann
Journal of Clinical Microbiology Sep 1998, 36 (9) 2557-2564; DOI: 10.1128/JCM.36.9.2557-2564.1998
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KEYWORDS

RNA, Ribosomal, 16S
RNA, Ribosomal, 23S
Yersinia
Yersinia Infections

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