Association of Mycobacterium avium subspecies paratuberculosis (MAP) with Crohn's disease (CD) and not with ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), has been vigorously debated in recent years. This theory has been strengthened by recent culture of MAP from breast milk, intestinal tissue and Blood from patients with active Crohn's disease. Culture of MAP from clinical samples remained challenging due to the fastidious nature of MAP including its lack of cell wall in infected patients. The advent of real time PCR has proven to be significant in infectious disease diagnostics. In this study, real time reverse transcriptase PCR (RT-PCR) assay based on targeting mRNA of the IS900 gene unique to MAP has been developed. All variables included in RNA isolation, cDNA synthesis and real time PCR amplification have been optimized. Oligonucleotide primers were designed to amplify 165 bp specific to MAP and the assay demonstrated sensitivity of 4 genomes per sample. In hope this real time RT-PCR may aid in the detection of viable MAP cells in Crohn's disease patients, a total of 45 clinical samples were analyzed. Portion of each sample was also subjected to 12 weeks culture followed by standard nested PCR analysis. The samples consisted of 17 cultures (originated from 13 CD, 1 UC and 3 NIBD subjects), 24 buffy coat blood (originated from 7 CD, 2 UC, 11 NIBD and 4 healthy subjects) and 4 intestinal biopsies from 2 CD patients. Real time RT-PCR detected viable MAP in 11/17 (65%) of iii suspected cultures compared to 12/17 (70%) by nested PCR including 77% and 84% from CD samples by both methods, respectively. Real time RT-PCR detected MAP RNA directly from 3/7 (42%) CD, 2/2 (100%) UC and 0/4 healthy controls similar to results following long term culture incubation and nested PCR analysis. Interestingly, real time RT-PCR detected viable MAP in 2/11 (13%) compared to 4/11 (26%) by culture and nested PCR in NIBD patients. For tissue samples, real time RT-PCR detected viable MAP in one CD patient with the culture outcome remains pending. This study clearly indicates that a 12-hr real time RT-PCR assay provided data that are similar to those from 12 weeks culture and nested PCR analysis. Consequently, use of real time In our laboratory, we previously demonstrated a possible downregulation in the Interferon-gamma receptor gene (IFNGR1) in patients with active Crohn's disease using microarray chip analysis. In this study, measurement of RNA by real time qRT-PCR indicated a possible downregulation in 5/6 CD patients compared to 0/12 controls. The preliminary data suggest that downregulation in INFGR1 gene, and the detection of viable MAP in CD patients provides yet the strongest evidence toward the linkage between MAP and CD etiology.