Tomaso Herbert, Jacob Daniela, Eickhoff Meike, Scholz Holger C, Al Dahouk Sascha, Kattar Mireille M, Reischl Udo, Plicka Helga, Olsen Jaran Strand, Nikkari Simo, Matero Pirjo, Beuret Christian, Ciammaruconi Andrea, Lista Florigio, Gala Jean-Luc, Broll Hermann, Appel Bernd, Sellek Cano Ricela E, Ybarra de Villavicencio Maria Del Carmen, Broekhuijsen Martien, Indra Alexander, Petersen Roger, Neubauer Heinrich
Bundeswehr Institute of Microbiology, Munich, Germany.
Clin Chem Lab Med. 2008;46(9):1239-44. doi: 10.1515/CCLM.2008.251.
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.
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.
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.
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.
鼠疫耶尔森菌是一种主要在啮齿动物及其跳蚤之间传播的人畜共患病细菌。传播给人类可导致腺鼠疫、肺鼠疫或败血性鼠疫,病死率很高。因此,快速可靠的诊断工具至关重要。本研究的目的是评估自行开发的用于鉴定鼠疫耶尔森菌的实时荧光定量PCR检测方法在不同实验室间的重复性。
将4份定量的鼠疫耶尔森菌DNA样本和2份空白样本盲法分发给14个实验室。为使操作标准化,提供了寡核苷酸,并使用相同的仪器平台和商业预混液。要求参与者报告其结果,包括循环阈值和熔解温度值。
所有参与实验室均能按照提供的方案进行实时荧光定量PCR检测,并正确鉴定出含有鼠疫耶尔森菌DNA的样本。参考实验室与参与实验室之间在循环阈值和熔解温度方面存在显著差异。然而,这并未对结果的判读产生不利影响。
我们的实时荧光定量PCR系统具有高度重复性,有潜力作为快速鉴定鼠疫耶尔森菌分离株的诊断工具的补充。需要进一步进行验证步骤,以确定临床样本的诊断准确性和预测价值。