Technical Abstract: Detection of Johne’s disease, an enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), has been impeded by the lack of rapid, reliable detection methods. The goal of this study was to optimize methodologies for obtaining, extracting and evaluating the concentration of M. paratuberculosis in manure from an infected dairy cow or in contaminated soil samples using a quantitative, real-time PCR (QRT-PCR) based analysis. Three different nucleic acid extraction techniques, the efficiency of direct versus indirect sample extraction, and sample pooling were assessed. The limit of detection was investigated by adding dilutions of M. paratuberculosis to soil. Results show that the highest yield of DNA (117 +/- 14 ng µl-1) and the highest copy number of the targeted M. paratuberculosis IS900 sequence (1.3 +/- 0.2 X 10^8 copies g-1) were obtained with DNA extracted from manure using the Fast® DNA Spin kit for soil. Pooling ten samples of M. paratuberculosis-contaminated soil improved the limit of detection ten fold. The limit of detection was between 20 and 115 M. paratuberculosis cells g-1 soil and detection was between 65% and 95% higher when samples were extracted directly using bead-beating than when using pre-treatment with cell extraction buffers. The final soil-sampling and extraction regime was applied for detection of M. paratuberculosis in pasture soil after the removal of a M. paratuberculosis culture positive dairy cow. M. paratuberculosis remained in the pasture soil for more than 200 days. Results from these studies suggest that DNA extraction method, sampling protocol and PCR conditions each critically influence the outcome and validity of the QRT-PCR analysis of M. paratuberculosis concentrations is environmental samples.