The objectives of this study were: to evaluate a novel fecal DNA extraction method, combined with a quantitative real-time PCR (QRTPCR) amplification method (VetAlertTM, Tetracore, Inc.) for the detection of MAP in bovine fecal samples from naturally infected cows, and 2) evaluate the use of the QRTPCR for confirmation of liquid culture of MAP.Fecal samples from multiple cattle shedding MAP at various levels were tested. The samples were processed with a DNA extraction and purification method which employed bead-beating and chaotropic solid phase extraction. Fecal DNA samples were then subjected to the QRTPCR amplification procedure, which is a real-time fluorescent probe hydrolysis assay. Shedding status was classified as negative, light, medium or heavy based on HEYM culture results. All of the heavy and moderate shedders were positive on QRTPCR. Approximately 30% of the samples from light shedders were positive. All of the culture-negative samples were negative on QRTPCR. The number of PCR cycles to reach positive detection threshold, which is related to the amount of DNA in the test sample, was significantly correlated to the number of colonies obtained on culture. Various DNA extraction procedures for MAP in liquid culture media were tested. Egg yolk added to liquid culture media appeared to inhibit the QRTPCR, and thus boiling or chaotropic solid phase extraction provided best results. All samples that flagged positive in the automated liquid culture machine were also detected as positive by QRTPCR.The DNA extraction procedure and quantitative real-time PCR assay show good sensitivity and specificity for the detection and quantification of MAP in bovine fecal samples and liquid culture samples.