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Journal of Clinical Microbiology, September 2005, p. 4744-4750, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4744-4750.2005

Development of a Nested PCR Method Targeting a Unique Multicopy Element, ISMap02, for Detection of Mycobacterium avium subsp. paratuberculosis in Fecal Samples

J. R. Stabel* and J. P. Bannantine

USDA-ARS, National Animal Disease Center, Bacterial Diseases of Livestock Research Unit, 2300 Dayton Rd., Ames, Iowa 50010

Received 22 December 2004/ Returned for modification 20 March 2005/ Accepted 13 May 2005

This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis 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 IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 102 CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 102 CFU/g for the IS900 element and 103 CFU/g for the ISMap02 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 M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 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 ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.

* Corresponding author. Mailing address: USDA-ARS, National Animal Disease Center, Bacterial Diseases of Livestock Research Unit, 2300 Dayton Rd., Ames, IA 50010. Phone: (515) 663-7304. Fax: (515) 663-7458. E-mail: jstabel{at}nadc.ars.usda.gov.

Journal of Clinical Microbiology, September 2005, p. 4744-4750, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4744-4750.2005




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