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Preliminary validation of real-time PCR assays for the identification of Yersinia pestis (Authors' personal document)

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Author: Tomaso, H. · Jacobs, D. · Eickhoff, M. · Scholz, H.C. · Dahouk, · Kattar, M.M. · Reischl, U. · Plicka, H. · Strand Olsen, J. · Nikkari, S. · Matero, P. · Beuret, C. · Ciammaruconi, A. · Lista, F. · Gala, J.-L. · Broll, H. · Appel, B. · Sellek Cano, R.E. · Ybarra de Villavicencio, M.d.C. · Broekhuijsen, M.P. · Indra, A. · Petersen, R. · Neubauer, H.
Source:Clinical Chemistry and Laboratory Medicine, 9, 46, 1239-1244
Identifier: 183518
doi: doi:10.1515/CCLM.2008.251
Keywords: Defence · Real-time PCR · Yersinia pestis · Article · Bacterium identification · Bacterium isolate · Controlled study · Diagnostic accuracy · Nonhuman · Nucleotide sequence · Priority journal · Real time polymerase chain reaction · Validation process · Yersinia pestis · Biological Warfare Agents · Laboratories · Polymerase Chain Reaction · Reproducibility of Results · Time Factors · Yersinia pestis · Bacteria (microorganisms) · Rodentia · Siphonaptera (fleas) · Yersinia pestis


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. © 2008 by Walter de Gruyter.