Resumo

PEREZ, I et al. Molecular identification of micobacteria in the tuberculous complex: standarization of a pcr method based on conserved sequences of the 16s-23s spacing region in the ribosomal dna operon. Rev. Soc. Ven. Microbiol. [online]. 2003, vol.23, n.1, pp.30-37. ISSN 1315-2556.

    In order to improve the diagnosis of tuberculosis, a methodology to identify mycobacteria belonging to the tuberculosis complex has been designed, based in sequence comparison of the 16S-23S region of the ribosomal DNA operon from 30 mycobacterial species. The method uses a combination of Polymerase Chain Reaction (PCR) with four primers (TTS, SS2, S1A and S1B), and digestion by restriction enzymes NheI and MscI. The technic was standarized using six mycobacterial isolates (M. tuberculosis, M. kansasii, M. fortuitum, M. smegmatis, M. marinum, M. terrae), obtaining amplification and digestion in all cases.     We conclude that the combination of PCR and digestion with restriction enzymes, represent a fast, simple, sensitive and specific method for identification of mycobacteria belonging to the tuberculosis complex (M. tuberculosis, M. bovis and M. africanum). However it is important to consider few recommendations to help decrease the unspecific amplification problems that appear during the study. One of the most important is the use of TTS-SS2 amplicon only as control for the presence and integrity of the bacterial DNA, and the combination of PCR and Nhe1 digestion only in the TTS-S1B fragment.

Palavras-chave : Micobacterias; tuberculosis; ADN; Mycobacterium tuberculosis.