Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: October 21, 2004
Publication Date: October 21, 2004
Citation: Stabel, J.R., Bannantine, J.P., Paustian, M. 2004. Development and Evaluation of a new PCR test for the detection of Mycobacterium avium subsp. paratuberculosis [abstract]. American Association of Veterinary Laboratory Diagnosticians. Technical Abstract: Paratuberculosis has become more widespread in both domestic and wild ruminants in the US and other parts of the world in recent years. Accurate and sensitive antemortem diagnosis of paratuberculosis is essential for implementation of national control programs. Fecal detection of M. paratuberculosis is the only methodology that can traverse both subclinical and clinical stages of disease. Yet fecal culture requires between 8 and 16 weeks for definitive results causing delays in management decisions to cull infected animals or purchase replacement animals. Nucleic acid detection methods using PCR have made rapid detection of M. paratuberculosis in fecal samples possible, reducing detection time to 2 to 3 days. Application of real-time PCR, nested PCR, and TaqMan PCR to detection assays for M. paratuberculosis have improved the sensitivity of detection and made it possible to perform semi-quantitative analyses. However, these tests have been based upon the amplification of the IS900 (multicopy) gene. This study describes the development of a nested PCR assay utilizing a unique gene for M. paratuberculosis (Map02) that is present in 6 copies in the genome. In addition, the sensitivity of this assay was compared to the detection sensitivity of the IS900 gene in both conventional and real-time PCR assays. Sensitivity of the PCR tests was evaluated using both naturally and experimentally infected samples. In addition, titration curves were generated using pure cultures of M. paratuberculosis and negative fecal samples spiked with known quantities of M. paratuberculosis. Specificity of the Map02 gene was evaluated by PCR of DNA from several isolates of M. paratuberculosis and M. avium subsp. avium, and DNA from M. fortuitum, M. scofulaceum, M. abcessan, M. smegmatis, and M. gordonea. Only M. paratuberculosis DNA was detected by the Map02 gene. Sensitivity of detection for the Map02 gene in either conventional or real-time PCR format determined using pure bacterial DNA was 100 fg DNA, compared to 10 fg DNA detected by the IS900 gene. Experimental spiking of a negative fecal sample followed by Map DNA extraction resulted in detection thresholds of 100 cfu/g for both IS900 and Map02 genes using a real-time PCR format but this sensitivity dropped 10-fold for both genes in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a higher sensitivity for the detection of Map DNA using the IS900 gene compared to the Map02 gene when it was used in a conventional PCR format. However, the real-time PCR format significantly improved detection levels of the Map02 gene. Using the real-time format detection of fecal samples a higher number of samples containing <10 cfu/g was possible compared to conventional PCR. In conclusion, the Map02 gene provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.