A novel real-time PCR assay for specific detection and quantification of Mycobacterium avium ssp. paratuberculosis in milk with the inherent possibility of differentiation between viable and dead cells
Abstract number: O242
Dzieciol M., Volgger P., Khol J., Baumgartner W., Wagner M., Hein I.
Objectives:Mycobacterium avium subsp. paratuberculosis (MAP) is known as the etiological agent of paratuberculosis (Johne's disease) in ruminants. Crohn's disease is an inflammatory gastrointestinal tract disease in humans, presenting with similar symptoms and pathological changes in the gut as Johne's disease in cattle. Therefore, it was suggested that MAP could be one of the etiologic factors of the disease. The aim of the present study was to develop a MAP-specific real-time PCR assay providing the additional possibility of detecting viable MAP.
Methods: A real-time PCR assay based on amplification of the specific Mptb52.16 target was designed including an internal amplification control to identify false negative results. The detection limit was established in artificially contaminated raw milk samples and the optimized assay applied to 96 naturally contaminated raw milk samples. The potential of the real-time PCR assay to detect viable MAP was explored by assessing expression of the Mptb52.16 target in raw milk samples and inoculated Dubos broth.
Results and Conclusions: The method showed 100% inclusivity and exclusivity when testing 11 MAP strains, 22 non-MAP mycobacteria, and 16 raw milk microflora strains. The detection limit in artificially contaminated raw milk was 2.42×101 MAP cells/ml milk. In a survey of naturally contaminated samples obtained from dairy herds with a known history of paratuberculosis, 47.8% pre-milk and 51.9% main milk samples tested positive. Real-time PCR-derived MAP-specific bacterial cell equivalents (BCE) ranged from 1×100 to 5.1×102 BCE/51.44 ml; the majority of samples had less than one BCE per ml milk. Expression of the chosen target was detected in artificially contaminated raw milk as well as inoculated Dubos broth, thus confirming the real-time PCR assay's potential to detect viable MAP cells.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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