The Study of PncA Gene Using PCR-RFLP and Allele-Specific PCR Methods in Distinguishing Mycobacterium Bovis from Mycobacterium Tuberculosis

Bovine tuberculosis is a chronic bacterial disease of cattle that occasionally affects human. A unique mutation at position 169 (C→G) in Mycobacterium bovis differentiates it from Mycobacterium tuberculosis. For the first time, PCR-RFLP method was used in this study to separate the two organisms. In this study, 98 tuberculosis patients were assessed using microscopic evaluations, culture and antibiogram. Then, nucleotide changes in position 169 were investigated using Allele-specific PCR and PCR-RFLP methods. Finally, the results were compared with spoligotyping results. Antibiogram revealed 28 subjects (28.57%) to be resistant and 54 (55.1%) to be sensitive to pyrazinamide while and 16 cases (16.23%) showed no growth. Of 28 resistant patients, 3 (3.1%) were diagnosed with M. Bovis using the classical methods. Using molecular methods, 95 (96.93%) out of 98 subjects were diagnosed with M. tuberculosis and 3 (3.06%) with M. bovis. Allele-specific PCR and PCR-RFLP matched with spoligotyping results. Our result showed the incidence of M. bovis infection to be higher in Iranian population (3.1%) than other studied cases (0.5-1%). In addition, we showed that Allele-specific PCR is the method of choice because it is faster and cheaper than PCR-RFLP. However, PCR-RFLP could be a proper alternative to prevent probable mismatches in Allele-specific PCR.