Stabel J R, Bannantine J P
USDA-ARS, National Animal Disease Center, Bacterial Diseases of Livestock Research Unit, Ames, IA 50010, USA.
J Clin Microbiol. 2005 Sep;43(9):4744-50. doi: 10.1128/JCM.43.9.4744-4750.2005.
This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis that is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 10(2) CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 10(2) CFU/g for the IS900 element and 10(3) CFU/g for the ISMap02 element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.
本研究描述了一种巢式PCR检测方法的开发，该方法使用一种独特的元件（ISMap02）来检测鸟分枝杆菌副结核亚种，该元件在基因组中以六个拷贝存在。此外，将该元件检测方法的灵敏度与传统PCR和实时PCR检测IS900元件的灵敏度进行了比较。通过对鸟分枝杆菌副结核亚种、鸟分枝杆菌亚种、偶然分枝杆菌、瘰疬分枝杆菌、草分枝杆菌、耻垢分枝杆菌和戈登分枝杆菌分离株提取的DNA进行PCR，评估了ISMap02元件的特异性。用ISMap02引物扩增后，仅能检测到鸟分枝杆菌副结核亚种的DNA。在纯细菌的系列滴定曲线中，传统或实时PCR形式下ISMap02元件的检测灵敏度低于100 fg DNA或10² CFU/ml。这些结果与IS900元件的结果相当。对阴性粪便样本进行实验性加样，随后提取鸟分枝杆菌副结核亚种DNA，使用实时PCR形式时，IS900元件的检测阈值为10² CFU/g，ISMap02元件的检测阈值为10³ CFU/g，但在传统PCR形式下，两种元件的灵敏度均下降了10倍。对自然感染动物的粪便样本进行分析表明，使用ISMap02元件检测鸟分枝杆菌副结核亚种DNA的灵敏度与使用IS900元件在传统PCR形式下的灵敏度相似。实时PCR形式提高了两种元件的检测水平，但提升幅度不大。总之，ISMap02元件作为一种诊断试剂，为用于检测副结核的PCR检测提供了一种非常灵敏且特异的替代方法。
