Cousins D V, Whittington R, Marsh I, Masters A, Evans R J, Kluver P
Animal Health Laboratories, Agriculture Western Australia, Locked Bag No. 4, Bentley Delivery Service, Bentley, WA Australia.
Mol Cell Probes. 1999 Dec;13(6):431-42. doi: 10.1006/mcpr.1999.0275.
PCR targeting the 5' end of IS 900 has been considered specific for identification of Mycobacterium avium subsp. paratuberculosis and is frequently applied to confirm the presence of this organism in the diagnosis of Johne's disease. IS 900 PCR has also been applied to studies of the aetiology of Crohn's disease. Mycobacterium spp. isolated from the faeces of 3 clinically normal animals in 2 Australian states appeared not to be M. paratuberculosis but were positive by IS 900 PCR. The isolates were characterized using mycobactin dependency, biochemical tests, IS 900 and 16 S rRNA sequencing and restriction fragment length polymorphism (RFLP) using IS 900 as probe. DNA sequencing confirmed that the isolates had between 71% and 79% homology with M. paratuberculosis in the region of IS 900 amplified, were most closely related to Mycobacterium scrofulaceum, and confirmed the usefulness of restriction enzyme analysis of amplified product to identify the false positive results. RFLP analysis with Bst Ell detected three to five copies of the IS 900 -like element in the isolates. These were located in molecular weight fragments that were clearly different to IS 900 in previously characterized strains of M. paratuberculosis. It is likely that these isolates are environmental mycobacteria. Southern blotting with an internal probe is unlikely to provide differentiation of M. paratuberculosis from these organisms. We recommend the adoption of restriction endonuclease analysis of IS 900 PCR product as a routine precaution to prevent the reporting of false positive IS 900 PCR results.
靶向IS 900 5'端的聚合酶链反应（PCR）被认为是副结核分枝杆菌鉴定的特异性方法，常用于诊断约内氏病时确认该菌的存在。IS 900 PCR也已应用于克罗恩病病因学的研究。从澳大利亚两个州的3只临床正常动物粪便中分离出的分枝杆菌似乎不是副结核分枝杆菌，但IS 900 PCR检测呈阳性。使用分枝杆菌素依赖性、生化试验、IS 900和16S rRNA测序以及以IS 900为探针的限制性片段长度多态性（RFLP）对分离株进行了鉴定。DNA测序证实，在扩增的IS 900区域，分离株与副结核分枝杆菌的同源性为71%至79%，与瘰疬分枝杆菌关系最为密切，并证实了对扩增产物进行限制性酶切分析以鉴定假阳性结果的有效性。用Bst Ell进行RFLP分析检测到分离株中有三到五个拷贝的IS 900样元件。这些元件位于分子量片段中，与先前鉴定的副结核分枝杆菌菌株中的IS 900明显不同。这些分离株可能是环境分枝杆菌。用内部探针进行Southern印迹不太可能区分副结核分枝杆菌和这些菌。我们建议采用对IS 900 PCR产物进行限制性内切酶分析作为常规预防措施，以防止报告IS 900 PCR假阳性结果。
