Al-Shamali M, Khan I, Al-Nakib B, Al-Hassan F, Mustafa A S
Dept. of Biochemistry, Faculty of Medicine, Kuwait University, Kuwait.
Scand J Gastroenterol. 1997 Aug;32(8):819-23. doi: 10.3109/00365529708996540.
Mycobacterium paratuberculosis is implicated as a possible cause of Crohn's disease. However, due to lack of an appropriate diagnostic method, this has been a subject of significant controversy. Our aim was therefore to develop a multiplex polymerase chain reaction (MPCR) for the detection of M. paratuberculosis DNA in Crohn's disease tissue.
Biopsy samples were collected by endoscopic forceps from terminal ileum, and genomic DNA was isolated. M. paratuberculosis-specific marker genes were amplified by using the present MPCR method.
Here we report a new MPCR for detection of M. paratuberculosis DNA in Crohn's disease tissue. In this technique two genetic markers, IS900 and a newly described specific marker of MP2, were amplified in a single tube simultaneously. The method was evaluated using biopsy specimens from 10 Crohn's disease patients, 6 ulcerative colitis patients, and 21 irritable bowel syndrome patients. The patients were characterized by using standard clinical and histologic observations. The present MPCR method could not detect M. paratuberculosis DNA in the biopsy specimens. However, the marker genes were amplified from the samples that were spiked with M. paratuberculosis before DNA extraction. The marker genes were also not detected in 10 closely related mycobacterial strains and human genomic DNA.
The present MPCR method is highly specific and can detect M. paratuberculosis DNA more reliably. These findings do not support an aetiologic role of M. paratuberculosis in Crohn's disease.
副结核分枝杆菌被认为可能是克罗恩病的病因之一。然而，由于缺乏合适的诊断方法，这一直是一个备受争议的话题。因此，我们的目的是开发一种多重聚合酶链反应（MPCR），用于检测克罗恩病组织中的副结核分枝杆菌DNA。
通过内镜钳从回肠末端采集活检样本，并分离基因组DNA。使用现有的MPCR方法扩增副结核分枝杆菌特异性标记基因。
在此我们报告一种用于检测克罗恩病组织中副结核分枝杆菌DNA的新型MPCR。在该技术中，两个遗传标记，即IS900和新描述的MP2特异性标记，在单个管中同时扩增。使用来自10例克罗恩病患者、6例溃疡性结肠炎患者和21例肠易激综合征患者的活检标本对该方法进行评估。通过标准临床和组织学观察对患者进行特征描述。现有的MPCR方法未能在活检标本中检测到副结核分枝杆菌DNA。然而，在DNA提取前加入副结核分枝杆菌的样本中扩增出了标记基因。在10株密切相关的分枝杆菌菌株和人类基因组DNA中也未检测到标记基因。
现有的MPCR方法具有高度特异性，能够更可靠地检测副结核分枝杆菌DNA。这些发现不支持副结核分枝杆菌在克罗恩病中的病因学作用。
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