Using on a phage display library expressing random 12-mer peptides, we isolated a peptide specific for the MptD protein of Mycobacterium avium subspecies paratuberculosis (MAP). The peptide had been synthesized without further modifications and designated as aMptD (Stratmann et al, Infect. Immun. 2004).To further investigate the specificity of the peptide aMptD, surface plasmon resonance (SPR) analysis was performed. For SPR analysis isolated membranes of MAP and M. avium were used as analytes and coupled to a gold-dextran matrix; aMptD and a random control peptide were injected as ligands. SPR analysis confirmed the specificity of aMptD for MAP. For further investigation of a potential diagnostic applicability of aMptD, paramagnetic beads were coated with aMptD. Milk samples were spiked with MAP and incubated overnight with the aMptD beads. After washing, the beads were resuspended in water and boiled. A MAP-specific PCR based on ISMav2 fragment was performed directly on the supernatant. Using this method, it was possible to reproducibly detect 10² MAP per ml milk. The specificity of the aMptD coated beads was confirmed by performing a competitive capture, using a 10³ fold excess of various mycobacterial species. Capture was followed by PCR using primers specific for the genus Mycobacteria. Via restriction enzyme digests of the resulting fragments, it was confirmed that only MAP had been captured. In order to show binding of aMptD to different isolates a capture of various MAP isolates (Type I and II) out of spiked milk was performed. Also, it was possible to detect MAP in individual and bulk milk samples. Based on these findings, we suggest the use of aMptD coated beads for the detection of MAP in milk in the microbiological routine laboratory