Mycobacterium paratuberculosis Detected by Nested PCR in Intestinal Granulomas Isolated by LCM in Cases of Crohns Disease

It has long been questioned whether Mycobacterium paratuberculosis plays a role in the aetiology of Crohn’s disease (1 ,2 ). Several methods of detection (serology, culture, and PCR)have been employed to prove or disprove a link, but without broad consensus to date (3 , 4 ). With granulomatous disease, it is reasonable to expect a (partially)causative infectious agent to be present in the granuloma, analogous to cases of M. tuberculosis (5 ). In this chapter we outline a method using Laser-Capture Microdissection (LCM)to isolate granulomata in cases of Crohn’s disease, and explain how to test for the presence of M. paratuberculosis using nested PCR. Formalin-fixed, paraffin-embedded tissue samples are examined using the LCM microscope. Granulomata are isolated and processed along with corresponding full-thickness samples. The DNA is harvested from all samples, and PCR is used to test the ability to amplify normal human genes—amplifying a 133-base-pair (bp)segment of the human adenomatous polyposis coli (APC) gene. To test for the presence of M. paratuberculosis , nested PCR amplifying a 193-bp and then a 155-bp internal fragment of the M. paratuberculosis IS900 region is used. This specific insertion sequence (IS)—IS900—is a multicopy DNA insertion element that is unique to the genome of M. paratuberculosis (6 ). Products are visualized by gel electrophoresis and staining with SYBR Green 1 nucleic acid gel stain. Tissue from a positive control, culture-positive specimen—taken from an animal infected with M. paratuberculosis —together with purified M. paratuberculosis bacterial isolates—are used as positive controls. Similarly treated samples of several types of non-Crohn’s granulomatous disorders are used as disease controls. The specificity of amplified PCR products can then be confirmed by restriction mapping or by direct sequencing.