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) may result from mycobacterial infection has been proposed. Among the atypical mycobacteria, Mycobacterium paratuberculosis has been often involved in the pathogenesis of CD and UC. The polymerase chain reaction (PCR) with a single primer pair from the nucleotide sequence of the 'Insertion Sequence' IS900 of M. paratuberculosis followed by a non-isotopic ELISA-like detection method of amplification products for the specific detection of M. paratuberculosis in human feces was developed. Fifteen (46.8%) of the 32 stool samples from patients with histologically confirmed CD, and nine (33.3%) of the 27 stool samples from patients 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 patients with other non-specific gastrointestinal diseases, and two from patients with colon cancer. Only one of these samples, namely from one 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 colon cancer allowed the mycobacterial growth. The amplified PCR products were identified by using a colorimetric detection procedure designed DNA Enzyme ImmunoAssay (DEIA), based on the hybridization of the denatured DNA with a non-radioactively labelled inter-primer specific oligonucleotide probe. Severe precautions were taken to exclude either the possible contamination among the samples or false-positive results. Our findings confirm other works in which M. paratuberculosis has been considered the putative etiologic agent responsible for CD and UC. In addition, the newly developed PCR-DEIA technique, revealing a higher sensitivity than cultural technique and being much more rapid, represents a useful tool for both epidemiological and therapeutic purposes. Copyright (C) 1998 Elsevier Science B.V.