A rapid, cheap and sensitive detection method of Mycobacterium avium subsp. paratuberculosis (MAP) in raw milk was needed for routine usage. We developed two duplex real time qPCR systems specific for MAP detection. These real time qPCR assays amplify the multicopy element IS900 for qualitative analysis and the single copy element F57 for quantitative analysis. Both assays incorporate an internal amplification control amplified with the same primers as the targets and the same probes are used in both assays. The specificity of the assays was confirmed by the testing of 6 different MAP isolates, 12 isolates of other mycobacteria or bacterial species and 4 different mammalian DNAs. The sensitivity of the developed assays and isolation efficiency were demonstrated through the analysis of raw milk samples artificially contaminated with MAP cells and with plasmids containing cloned fragments of the targets (IS900 and F57). The developed assays for milk analysis were applied to samples from one farm with two faecal shedding cows. Three hundred and forty five individual milk samples were tested by real time qPCR assays and by cultivation. Hundred and eleven (32.5%) individual milk samples were positive by the real time qPCR, no milk sample was culture positive. The spread of MAP in individual, tank and bulk tank milk samples was also monitored.

Veterinary Research Institute Hudcova 70 621 00 Brno Czech Republic

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