Clinical Chemistry and Laboratory Medicine (CCLM)
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Preliminary validation of real-time PCR assays for the identification of Yersinia pestis
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
Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 46, Issue 9, Pages 1239–1244, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2008.251, July 2008
- 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.
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