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Evaluation of a duplex PCR assay for the identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria

Abstract:

The recent resurgence of tuberculosis at world wide level demands the design of rapid, sensitive and specific diagnostic strategies for early detection of pathogen. It was evaluated a duplex PCR assay for the detection and differentiation of Mycobacterium tuberculosis complex (TB) and nontuberculous mycobacteria (NTB). In a single amplification reaction, two primer sets were included and their annealing temperatures were standardized. In order to detect any mycobacteria (TB and NTB), a set of primers directed to the conserved region of the 16S rRNA gene were used. In order to identify mycobacteria of the M. tuberculosis complex, another primers were designed from the open reading frame Rv0577 sequence from the genome M. tuberculosis reference strain H37Rv. To validate the test, 50 clinical isolates of mycobacteria were analyzed, including mycobacteria of the TB complex and NTB mycobacteria. In all the species of mycobacteria tested with oligonucleótidos of 16S rRNA, a PCR product of 543 pb was observed, confirming that the primers set used were specific-genus. The reaction with Rv0577 primers set specifically and consistently amplified an additional PCR product of 786 pb in M. tuberculosis, M. bovis and M. bovis BCG, the strains of the TB complex analyzed in this study, whereas none of the NTB strain showed a PCR fragment. Given the sensitivity and specificity of the primer sets used, this duplex PCR assay can contribute to the diagnosis of micobacteriosis and may be applied to identify mycobacteria of the M. tuberculosis complex and nontuberculous mycobacteria in clinical samples from humans and animals.