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
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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