Molecular based detection strategies for Mycobacterium avium subsp. paratuberculosis (MAP) offer the benefit of speed over the traditional culturing methods. In this study we attempted to evaluate and optimise a real-time PCR approach which may be suitable for detecting and quantifying MAP in culture using SYBR green and the highly characterised P90 / P91 primers. All parameters of the PCR were carefully adjusted including Mg concentration, primer concentration and annealing temperature After optimising the assay, fluorescence readings for each sample per PCR cycle were recorded to determine the sensitivity of the reaction and sophisticated melting curve analysis was used to determine specificity. This method was tested using a variety of templates including purified PCR products, isolated MAP DNA, pure colonies or liquid culture sources. The sensitivity of this assay was as follows: purified template - 20 copies, isolated DNA - 50fg, MAP in broth - 25 cells. Furthermore, this assay was found to be specific for MAP even in the presence of heavily contaminated samples, as determined by the presence of an identifiable melting peak at 92.2ºC. Two critical factors in optimising the success of this assay were primer (0.05µM) and Mg. concentrations (3mM). By adapting this assay it was found that it could be used to accurately enumerate the numbers of MAP cells growing in broth when compared with pre-determined standards. This application is significant as it may present researchers with an alternative or confirmatory method to counting colonies on slopes, or measuring growth in radiological media. In conclusion we have optimised a sensitive and specific real-time PCR assay for MAP which is effective using a variety of templates, and may be useful to researchers as a rapid means of detecting and enumerating MAP in culture