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Licensed Unlicensed Requires Authentication Published by De Gruyter July 21, 2008

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

  • Herbert Tomaso , Daniela Jacob , Meike Eickhoff , Holger C. Scholz , Sascha Al Dahouk , Mireille M. Kattar , Udo Reischl , Helga Plicka , Jaran Strand Olsen , Simo Nikkari , Pirjo Matero , Christian Beuret , Andrea Ciammaruconi , Florigio Lista , Jean-Luc Gala , Hermann Broll , Bernd Appel , Ricela E. Sellek Cano , Maria del Carmen Ybarra de Villavicencio , Martien Broekhuijsen , Alexander Indra , Roger Petersen and Heinrich Neubauer


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.

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

Received: 2008-3-12
Accepted: 2008-5-12
Published Online: 2008-07-21
Published in Print: 2008-09-01

©2008 by Walter de Gruyter Berlin New York

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