Development of a Nested PCR Method Targeting a Unique Multicopy Element, ISMap02, for Detection of Mycobacterium aviumsubsp. paratuberculosisin Fecal Samples

ABSTRACTThis study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium aviumsubsp. paratuberculosisthat is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900element in both conventional and real-time PCR assays. The specificity of the ISMap02element was evaluated by PCR of the DNA extracted from isolates of M. aviumsubsp. paratuberculosisand M. aviumsubsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. aviumsubsp. paratuberculosisDNA was detectable after amplification with the ISMap02primers. The sensitivity of detection for the ISMap02element in either a conventional or a real-time PCR format was less than 100 fg DNA or 102CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900element. Experimental spiking of a negative fecal sample followed by M. aviumsubsp. paratuberculosisDNA extraction resulted in detection thresholds of 102CFU/g for the IS900element and 103CFU/g for the ISMap02element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. aviumsubsp. paratuberculosisDNA by use of the ISMap02element similar to that achieved by use of the IS900element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.