An mph-64 flanking sequence specific for Mycobacterium bovis

FISANOTTI J.C.; ROMANO M; A. ALITO; BIGI F; A. CATALDI

RESEARCH IN MICROBIOLOGY

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 1997 vol. 148 p. 427 - 435

0923-2508

A clone carrying a plasmid with the mp6-64 gene and 3’ flanking sequences (plasmidpMBA122) was detected during the screening of a Mycobacferium bowis genomiclibrary with sera from infected cattle. When the pMBA122 insert was used as a probe inSouthern blots against /V&digested mycobacterial DNA, it distinguished the differentM, tuberculosis complex species. This probe hybridized with a 7-kb band in M. tuberculosis,a 5-kb band in M. bovis and a 3-kb band in M. tuberculosis complex strains fromwild seals. Smal genomic digestions enabled us to locate this polymorphic regiondownstream of the mph-64 gene. In order to clone this particular region, we designeda pair of PCR primers. Unexpectedly, these primers amplified only M. bovis DNA; noamplification was seen in M. tuberculosis DNA. When the annealing temperature waslowered from 70 to 55X, an amplification product of the same size was obtained withM, tuberculosis. This product was cloned and sequenced, and showed partial homologyto the M. bovis amplified fragment. Therefore, this region comprises M. bovissequences with a lower homology with M. tuberculosis than other comparedsequences. This suggests that a more precise differentiation method at the species levelfor the M. tuberculosis complex could be achieved using PCR directed to this region.