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Clinical Chemistry and Laboratory Medicine (CCLM)

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Preliminary validation of real-time PCR assays for the identification of Yersinia pestis

Herbert Tomaso1 / Daniela Jacob2 / Meike Eickhoff3 / Holger C. Scholz4 / Sascha Al Dahouk5 / Mireille M. Kattar6 / Udo Reischl7 / Helga Plicka8 / Jaran Strand Olsen9 / Simo Nikkari10 / Pirjo Matero11 / Christian Beuret12 / Andrea Ciammaruconi13 / Florigio Lista14 / Jean-Luc Gala15 / Hermann Broll16 / Bernd Appel17 / Ricela E. Sellek Cano18 / Maria del Carmen Ybarra de Villavicencio19 / Martien Broekhuijsen20 / Alexander Indra21 / Roger Petersen22 / Heinrich Neubauer23

1Bundeswehr Institute of Microbiology, Munich, Germany

2Robert Koch-Institut, Zentrum für Biologische Sicherheit, Hochpathogene mikrobielle Erreger, Berlin, Germany

3QIAGEN Hamburg GmbH, Hamburg, Germany

4Bundeswehr Institute of Microbiology, Munich, Germany

5Department of Internal Medicine III, RWTH Aachen University, Aachen, Germany

6Molecular Infectious Diseases Diagnostics, Department of Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon

7Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany

8BMLV/RD-ARWT, ABCUT, Mödling, Austria

9Norwegian Defense Research Establishment, Division for Protection, Kjeller, Norway

10Center for Biothreat Preparedness, and BC-Defense and Environmental Health Unit, Center for Military Medicine, Helsinki, Finland

11Center for Biothreat Preparedness, and BC-Defense and Environmental Health Unit, Center for Military Medicine, Helsinki, Finland

12Biology Spiez Laboratory, Federal Department of Defense, Civil Protection and Sports, Federal Office for Civil Protection, Spiez, Switzerland

13Health Corps, Italian Army, Histology and Molecular Biology Section, Army Medical and Veterinary Research Center, Roma, Italy

14Health Corps, Italian Army, Histology and Molecular Biology Section, Army Medical and Veterinary Research Center, Roma, Italy

15Center for Applied Molecular Technologies, Defense Laboratories Department, Belgian Armed Forces, Brussels, Belgium

16Bundesinstitut für Risikobewertung, Berlin, Germany

17Bundesinstitut für Risikobewertung, Berlin, Germany

18Unidad Biológica-NBQ, Fábrica Nacional “La Marañosa”, Spain

19Unidad Biológica-NBQ, Fábrica Nacional “La Marañosa”, Spain

20TNO Defense, Security and Safety, Rijswijk, The Netherlands

21AGES – Institut für medizinische Mikrobiologie und Hygiene, Wien, Austria

22TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany

23Friedrich Loeffler Institut, Jena, Germany

Corresponding author: Dr. Herbert Tomaso, Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, 80937 Munich, Germany Phone: +49-89-3168-3933, Fax: +49-89-3168-3292,

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 46, Issue 9, Pages 1239–1244, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2008.251, July 2008

Publication History

Published Online:


Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis.

Methods: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values.

Results: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results.

Conclusions: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples.

Clin Chem Lab Med 2008;46:1239–44.

Keywords: Yersinia pestis; real-time PCR

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