DETECTION OF MYCOBACTERIUM-PARATUBERCULOSIS IN STOOL SAMPLES OF PATIENTS WITH INFLAMMATORY BOWEL-DISEASE BY IS900-BASED PCR AND COLORIMETRIC DETECTION OF AMPLIFIED DNA

The hypothesis that Crohn's disease (CD) and ulcerative colitis (UC) m ay result from mycobacterial infection has been proposed. Among the at ypical mycobacteria, Mycobacterium paratuberculosis has been often inv olved in the pathogenesis of CD and UC, The polymerase chain reaction (PCR) with a single primer pair from the nucleotide sequence of the 'I nsertion Sequence' IS900 of M. paratuberculosis followed by a non-isot opic ELISA-like detection method of amplification products for the spe cific detection of M. paratuberculosis in human feces was developed. F ifteen (46.8%) of the 32 stool samples from patients with histological ly confirmed CD, and nine (33.3%) of the 27 stool samples from patient s with UC had a 229-bp fragment of M. paratuberculosis DNA detected by ethidium bromide agarose gel electrophoresis. Of the 41 stool samples used as negative control, 30 were from healthy subjects, nine from pa tients with other non-specific gastrointestinal diseases, and two from patients with colon cancer. Only one of these samples, namely from on e of the patients with colon cancer, was positive by PCR. With regard to cultural technique, eight stool samples from patients with CD, five samples from patients with UC and one sample from a patient with colo n cancer allowed the mycobacterial growth. The amplified PCR products were identified by using a colorimetric detection procedure designed D NA Enzyme ImmunoAssay (DEIA), based on the hybridization of the denatu red DNA with a non-radioactively labelled inter-primer specific oligon ucleotide probe. Severe precautions were taken to exclude either the p ossible contamination among the samples or false-positive results. Our findings confirm other works in which M. paratuberculosis has been co nsidered the putative etiologic agent responsible for CD and UC. In ad dition, the newly developed PCR-DEIA technique, revealing a higher sen sitivity than cultural technique and being much more rapid, represents a useful tool for both epidemiological and therapeutic purposes. (C) 1998 Elsevier Science B.V.