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Title:   Author  item
                                            Thacker, Tyleritem
                                            HARRIS, BETH - USDA-APHISitem
                                            Palmer, Mitchellitem
                                            Waters, Wade
  Submitted to: Meeting Abstract
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 10/18/2007
                                    Publication  Date: 10/18/2007
                                    Citation: Thacker, T.C., Harris, B., Palmer, M.V., Waters, W.R. 2007. Improved Specificity for Detection of Mycobacterium bovis in Fresh Tissue Using IS6110 Real-Time PCR [abstract].Interpretive Summary:
                                Technical Abstract: Introduction: Current accepted methods of detecting Mycobacterium bovis (M. bovis) in fresh tissues requires months for definitive culture results. It has been proposed that direct detection of M. bovis DNA in fresh tissues could provide more rapid results. These data would complement the current method of PCR detection of M. bovis in fixed tissues. One of the most significant impediments to the development of this technique is differentiation of M. bovis from environmental mycobacteria. PCR primers that amplify regions of the IS6110 region of the genome have been developed to differentiate members of the Mycobacterium tuberculosis complex from environmental mycobacteria. Currently primers developed by Eisenach et al. (J Inf Dis 161:977) named IS6110 are the most widely used in veterinary diagnostic applications. Another primer pair that, in preliminary experiments, was more sensitive in our hands was IS41/43 developed by Plikaytis et al. (Mol Cell Probes 5:215). To evaluate specificity of these primer pairs, mycobacteria reference strains were obtained from ATCC. 
Materials and Methods: DNA was isolated from the following mycobacteria; M. smegmatis, M. terrae, M. goodii, M. fortuitum, M. kansasii, M. avium ssp. paratuberculosis, M. wolinskyi, M. simiae, M. peregrinum, M. intracellulare, M. chelonae, M. avium and M. bovis. Previously published primers IS41/43 and IS6110 were assayed for detection. PCR positive samples were sequenced using standard techniques. New primers and a Taqman probe were designed based on sequences obtained from sequencing this region of each genome using Primer3 v0.3 (frodo.wi.mit.edu/primer3/). Real-time PCR was carried out using Taqman Master Mix (ABI, Foster City, CA) according the manufacturers directions with 0.4 uM of each primer (IS6110_T) and 0.2 uM probe (IS6110_T_probe). To test these new primers and probe, tissue samples from 48 cattle from a naturally infected herd were tested for M. bovis by real-time PCR. 
Results: The IS41/43 primer pair produced positive results with the following mycobacteria; M. terrae, M. goodii, M. fortuitum, M. wolinskyi and M. simiae. While the IS6110 primer pair generated positive PCR results from M. terrae, M. goodii, M. wolinskyi, M. peregrinum and M. chelonae. The IS6110 region of these genomes was sequenced and real-time primers and probe developed. Using the newly designed primers in a real-time PCR format these primers improved specificity by only detecting M. bovis, M. bovis BCG and M. wolinskyi. Based on sequencing data M. wolinskyi’s PCR product was 100% identical to that of M. bovis. 
To test the ability of the improved PCR primers and probe tissue samples from a naturally infected herd were tested for M. bovis infection. Of these samples 30 were M. bovis culture positive and 18 were M. bovis culture negative. We detected 20 of the 30 culture positive samples. None of the M. bovis culture negative samples were PCR positive using this assay. M. smegmatis was cultured from several of the M. bovis culture negative samples. These samples were PCR negative in this assay. Using this real-time assay, fewer false positives occur when assaying tissues from animals infected with mycobacteria other than M. bovis. Inclusion of this assay in the diagnostic scheme would provide rapid evidence of M. bovis infection in suspect animals.


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