Newly developed primers for the detection of Mycobacterium avium subspecies paratuberculosis

Recent publications reported the existence of IS900 like sequences in mycobacteria different from Mycobacterium avium subspecies paratuberculosis (Map). The primers used for IS900 detection of Map have amplified these sequences causing false positive results. In this study, we have developed two new PCR assays for the detection of Map. The first assay is based on the IS900 sequence using primers different from the ones previously reported, the second assay on the f57 sequence. The specificity of the tests was checked by analysis of 190 mycobacterial isolates (74 Map and 116 non-Map isolates). All Map strains were positive and all non-Map strains were negative. Serial dilutions of Map bacteria were used to assess the sensitivity of the assays. We achieved a sensitivity of 1CFU per PCR for both assays. In addition, a PCR-simulating computer programme was used to evaluate the specificity of the new IS900 primers. The combination of the two PCR assays has proven to be useful for the identification of Map but validation on a large range of clinical samples still needs to be done.