A multiplex polymerase chain reaction assay for the detection of Mycobacterium paratuberculosis DNA in Crohn's disease tissue

Background: 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. Methods: 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. Results: 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. Conclusions: 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.