Diagnosis of bovine tuberculosis by PCR and genotyping of Mycobacterium bovis by spoligotyping

In this study, the presence of Mycobacterium bovis DNA was investigated by Real-Time PCR in 72 tissue samples (36 of which were lymph nodes and 36 were blood samples) from 36 cows, suspected of having bovine tuberculosis, slaughtered at abattoirs in Ankara. M. bovis DNAs were genotyped by spoligotyping method. For this reason, bovine blood and lymph node tissues were spiked with M. bovis AN/5 strain and were used for PCR optimization. PCR optimization was done in Light Cycler (performing Real-Time Fluorescent detection) system. Total PCR time was found to be 40 min. Amplicons were detected to lose their fluorescent densities (in other words they were totally denatured) at 92.176C. This situation was specific for the region specifically amplified by the primers DRa and DRb. PCR sensitivity was determined as 6 cfu/2 ul = 3 cfu/ul. 9 M. bovis DNA from 36 lymph nodes out of 72 tissues samples were amplified by PCR and these were detected by ELISA. No amplification was recorded from blood samples. Spoligotyping method was performed in microplates. 43 different oligonucleotides coated in microplate wells were hybridized with 9 PCR amplicons. As a result, 3 different spoligotypes were detected in 9 M. bovis DNA samples.