Nucleotide fragment of DNA, whose nucleotide sequence is included in the gene of the species of the genus Mycobacterium, coding for the 65-kD mycobacterial antigen, containing regions which are homologous in practically all species of the genus Mycobacterium, and at least one species-specific variable region, characterized in that said fragment is chosen from fragments whose nucleotide sequences possess at least 70% homology, and preferably at least 85% homology, with a predetermined sequence or its complementary sequence, said predetermined sequence beginning at nucleotide 438 and ending at nucleotide 751 of said gene coding for said antigen of all species of mycobacteria except for the species M. tuberculosis, M. bovis BCG, M. avium, M. paratuberculosis, M. fortuitum, M. malmoense, M. leprae, M. kansaii and M. marinurn.