title: Preliminary validation of real-time PCR assays for the identification of Yersinia pestis.
abstract: 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.
authors.affiliation: Bundeswehr Institute of Microbiology, Munich, Germany. herbert.tomaso@web.de
authors.forename: Herbert
authors.initials: H
authors.lastname: Tomaso
authors.forename: Daniela
authors.initials: D
authors.lastname: Jacob
authors.forename: Meike
authors.initials: M
authors.lastname: Eickhoff
authors.forename: Holger C
authors.initials: HC
authors.lastname: Scholz
authors.forename: Sascha
authors.initials: S
authors.lastname: Al Dahouk
authors.forename: Mireille M
authors.initials: MM
authors.lastname: Kattar
authors.forename: Udo
authors.initials: U
authors.lastname: Reischl
authors.forename: Helga
authors.initials: H
authors.lastname: Plicka
authors.forename: Jaran Strand
authors.initials: JS
authors.lastname: Olsen
authors.forename: Simo
authors.initials: S
authors.lastname: Nikkari
authors.forename: Pirjo
authors.initials: P
authors.lastname: Matero
authors.forename: Christian
authors.initials: C
authors.lastname: Beuret
authors.forename: Andrea
authors.initials: A
authors.lastname: Ciammaruconi
authors.forename: Florigio
authors.initials: F
authors.lastname: Lista
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Gala
authors.forename: Hermann
authors.initials: H
authors.lastname: Broll
authors.forename: Bernd
authors.initials: B
authors.lastname: Appel
authors.forename: Ricela E
authors.initials: RE
authors.lastname: Sellek Cano
authors.forename: Maria Del Carmen
authors.initials: Mdel C
authors.lastname: Ybarra de Villavicencio
authors.forename: Martien
authors.initials: M
authors.lastname: Broekhuijsen
authors.forename: Alexander
authors.initials: A
authors.lastname: Indra
authors.forename: Roger
authors.initials: R
authors.lastname: Petersen
authors.forename: Heinrich
authors.initials: H
authors.lastname: Neubauer
mesh_terms: D054045:Biological Warfare Agents; D007753:Laboratories; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D015203:Reproducibility of Results; D013997:Time Factors; D015010:Yersinia pestis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D054045:Biological Warfare Agents