Development of a Nested PCR Method Targeting a Unique Multicopy Element, ISMap<i>02</i>, for Detection of<i>Mycobacterium avium</i>subsp.<i>paratuberculosis</i>in Fecal Samples

<jats:title>ABSTRACT</jats:title><jats:p>This study describes the development of a nested PCR assay that uses a unique element (ISMap<jats:italic>02</jats:italic>) for<jats:italic>Mycobacterium avium</jats:italic>subsp.<jats:italic>paratuberculosis</jats:italic>that 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 IS<jats:italic>900</jats:italic>element in both conventional and real-time PCR assays. The specificity of the ISMap<jats:italic>02</jats:italic>element was evaluated by PCR of the DNA extracted from isolates of<jats:italic>M. avium</jats:italic>subsp.<jats:italic>paratuberculosis</jats:italic>and<jats:italic>M. avium</jats:italic>subsp.<jats:italic>avium</jats:italic>, as well as DNA from<jats:italic>M. fortuitum</jats:italic>,<jats:italic>M. scofulaceum</jats:italic>,<jats:italic>M. phlei</jats:italic>,<jats:italic>M. smegmatis</jats:italic>, and<jats:italic>M. gordonae</jats:italic>. Only<jats:italic>M. avium</jats:italic>subsp.<jats:italic>paratuberculosis</jats:italic>DNA was detectable after amplification with the ISMap<jats:italic>02</jats:italic>primers. The sensitivity of detection for the ISMap<jats:italic>02</jats:italic>element in either a conventional or a real-time PCR format was less than 100 fg DNA or 10<jats:sup>2</jats:sup>CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS<jats:italic>900</jats:italic>element. Experimental spiking of a negative fecal sample followed by<jats:italic>M. avium</jats:italic>subsp.<jats:italic>paratuberculosis</jats:italic>DNA extraction resulted in detection thresholds of 10<jats:sup>2</jats:sup>CFU/g for the IS<jats:italic>900</jats:italic>element and 10<jats:sup>3</jats:sup>CFU/g for the ISMap<jats:italic>02</jats:italic>element 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<jats:italic>M. avium</jats:italic>subsp.<jats:italic>paratuberculosis</jats:italic>DNA by use of the ISMap<jats:italic>02</jats:italic>element similar to that achieved by use of the IS<jats:italic>900</jats:italic>element 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 ISMap<jats:italic>02</jats:italic>element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.</jats:p>