الفهرس 
SUPERVISION SHEETOnly 14 pages are availabe for public view
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Abstract
Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical-based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. A novel multiplex real-time PCR assay was developed and applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 4 other bacterial species such as Escherichia coli . The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR primers WHO recommended as the gold standard method, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA till 2 DNA copies. Consequently, this multiplex real-time PCR assay is a useful diagnostic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis.