Global Veterinaria 13 (5): 794-800, 2014 ISSN 1992-6197 © IDOSI Publications, 2014 DOI: 10.5829/idosi.gv.2014.13.05.86179 Corresponding Author: Khaled Al Amry, Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. 794 Detection and Differentiation between Mycobacterium bovis and Mycobacterium tuberculosis in Cattle Milk and Lymph Nodes Using Multiplex Real-Time PCR Suzan A. Mohamed, Kh. F. Mohamed, M.G. Aggour, Hanaa A. Ahmed and S.A. Selim 1 2 3 4 2 Tuberculosis Unit, Bacteriology Department, Animal Health Research Institute, Dokki, Giza, Egypt 1 Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt 2 Biotechnology Department, Animal Health Research Institute, Dokki, Giza, Egypt 3 Genome Unit, Animal Health Research Institute, Dokki, Giza, Egypt 4 Abstract: A novel multiplex real-time PCR assay was developed and applied directly to biological samples with evidence of bTB in order 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 present 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 3 other bacterial species such as: Escherichia coli, shigella Spp., salmonella Spp. in cattle milk and lymph nodes. The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. 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 as the gold standard, the relative accuracy of real-time PCR was 99.1% and the relative specificity was 100%.The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 2 DNA copies per PCR reaction. 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, as well as the epidemiologic surveillance of animals slaughtered in abattoir. Key words: Real-Time PCR Mycobacterium bovis Mycobacterium tuberculosis Complex IS1081 INTRODUCTION Because of the slow growth rate of Mycobacterium Mycobacterium bovis and closely associated susceptibility testing of this organism and other clinically acid-fast bacilli cause diseases in humans. Epidemiologic important mycobacteria can take several weeks or longer. investigations reveal that the organism may be ingested During the past several years, many molecular methods or inhaled. Extra pulmonary lesions may occur have been developed for direct detection, species associated with the consumption of infected milk, identification and drug susceptibility testing of even though with the practice of boiling milk and the mycobacteria. These methods can potentially reduce the growth of milk pasteurization plants all over the world. diagnostic time from weeks to days [3]. Strikingly, the The digestive route of infection has become less genome sequence of M. bovis is > 99.95% identical at important. On the other hand, airborne infection continues the nucleotide level to that of M. tuberculosis, showing to occur among meat industry and slaughterhouse collinearity and no evidence of extensive translocations, workers, in regions where the infection in cattle is still duplications or inversions. But deletion of genetic prevalent [1]. Major grouping of Mycobacterium information that has led to a reduced genome size, tuberculosis complex are: M. tuberculosis, M. bovis, M. revealed 11 deletions from the genome of M. bovis, microti and M. africanum [2]. ranging in size from ˜1 to 12.7 kb. Surprisingly, tuberculosis, isolation, identification and drug