Preliminary validation of real-time PCR assays for the identification of 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.
Tomaso
Herbert
H
Bundeswehr Institute of Microbiology, Munich, Germany. herbert.tomaso@web.de
Jacob
Daniela
D
Eickhoff
Meike
M
Scholz
Holger C
HC
Al Dahouk
Sascha
S
Kattar
Mireille M
MM
Reischl
Udo
U
Plicka
Helga
H
Olsen
Jaran Strand
JS
Nikkari
Simo
S
Matero
Pirjo
P
Beuret
Christian
C
Ciammaruconi
Andrea
A
Lista
Florigio
F
Gala
Jean-Luc
JL
Broll
Hermann
H
Appel
Bernd
B
Sellek Cano
Ricela E
RE
Ybarra de Villavicencio
Maria Del Carmen
Mdel C
Broekhuijsen
Martien
M
Indra
Alexander
A
Petersen
Roger
R
Neubauer
Heinrich
H
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*
D054045:Biological Warfare Agents