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Journal of Clinical Microbiology, September 1998, p. 2557-2564, Vol. 36, No. 9
Max-von-Pettenkofer-Institut für
Hygiene und Medizinische Mikrobiologie,
Received 10 April 1998/Returned for modification 2 June
1998/Accepted 19 June 1998
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 of
Yersinia 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 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 pathogenic Yersinia species (in particular Y. pestis and
Y. pseudotuberculosis) was developed. The applicability of
this technique was demonstrated by detection of Y. pestis
and Y. pseudotuberculosis in spiked throat and stool
samples, respectively. These probes were also capable of identifying
Y. enterocolitica within cryosections of experimentally
infected mouse tissue by the use of confocal laser scanning microscopy.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Development of rRNA-Targeted PCR and In Situ
Hybridization with Fluorescently Labelled Oligonucleotides for
Detection of Yersinia Species
*
Corresponding author. Mailing address: Max von
Pettenkofer Institut, Ludwig Maximilians Universität,
Pettenkoferstr. 9a, D-80336 Munich, Germany. Phone: 49 (89)
5160-5200/5201. Fax: 49 (89) 5160-5202. E-mail:
sekretariat{at}m3401.mpk.med.uni-muenchen.de.
Journal of Clinical Microbiology, September 1998, p. 2557-2564, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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