Ikonomopoulos John, Gazouli Maria, Pavlik Ivo, Bartos Milan, Zacharatos Panayotis, Xylouri Eftixia, Papalambros Efstathios, Gorgoulis Vassilis
Deparment of Anatomy-Physiology, Faculty of Animal Science, Agricultural University of Athens, Iera Odos 75, Botanikos 11855, Greece.
J Microbiol Methods. 2004 Mar;56(3):315-21. doi: 10.1016/j.mimet.2003.10.016.
Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.
鸟分枝杆菌副结核亚种（MAP）可在多种动物物种，尤其是反刍动物中引发一种非常严重且往往致命的疾病，即副结核病。最近，它还被认为与人的炎症性肠病发病机制有关。本研究的目的是开发一种分子方法，用于从动物源组织样本中常规检测和鉴定MAP。所提出的检测方法必须在性能、成本和高重复性之间达到最佳平衡。为实现这一目标，希腊和捷克共和国的三个实验室分别承担了一项研究的不同部分，该研究涉及评估用于MAP检测的DNA提取程序和PCR检测方法。对于DNA提取，我们使用了一种内部方法和一种商业方法；对于PCR，我们评估了许多不同的检测方法，首先通过扩展的GenBank数据库搜索来评估引物特异性。基于这些结果，我们选择评估针对IS900元件不同基因组区域的单管巢式PCR、两管巢式PCR和单重PCR检测方法。这四种方法应用于阳性和阴性对照样本，这些样本包括从患有副结核病的牛和感染鸟分枝杆菌鸟亚种的鸡中采集的纯细菌培养物以及福尔马林固定石蜡包埋（FFPE）组织样本。基于可靠性和成本标准，表现最佳的程序是单管巢式PCR检测方法与内部DNA提取方法相结合。三个合作实验室获得的结果一致性表明，即使在不同实验室条件下，所提出的检测方法也具有可靠性。
