Diferenciação de micobactérias por PCR multiplex Differentiation of micobacteria by multiplex PCR

• Published in: Revista da Sociedade Brasileira de Medicina Tropical
• Main Authors: Diogo da Rocha Poroca, Andrea Santos Lima, Juliana Falcão de Araújo Lima, Heidi Lacerda Alves da Cruz, Rosana de Albuquerque Montenegro, Fábio Lopes de Melo, Haiana Charifker Schindler, Lílian Maria Lapa Montenegro
• Format: Article in Journal/Newspaper
• Language: English
• Published: Sociedade Brasileira de Medicina Tropical (SBMT) 2009
• Subjects: Micobactérias; PCR multiplex; IS6110; dnaJ; hsp65; Mycobacteria; Multiplex PCR; Arctic medicine. Tropical medicine; RC955-962; Arctic
• Online Access: https://doi.org/10.1590/S0037-86822009000600020; https://doaj.org/article/f5bd222fefa54ec0857b721150601217

O trabalho visou à otimização de um método baseado na reação em cadeia da polimerase multiplex - para diferenciação de micobactérias de interesse para a saúde pública. A PCR Multiplex baseou-se na amplificação simultânea do genehsp65, presente em todo gênero Mycobacterium, do gene dnaJ, presente apenas em Mycobacterium tuberculosis e Mycobacterium avium e da sequência de inserção IS6110 presente no complexo Mycobacterium tuberculosis, gerando amplicons de 165pb, 365pb e 541pb, respectivamente. O limite de detecção foi de 1fg para o alvo hsp65, 100pg para o dnaJ e 0,1fg para o IS6110. A PCR multiplex detectou até 100pg de DNA de Mycobacterium tuberculosis. O sistema demonstrou ser específico e sensível na detecção de Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium e Mycobacterium smegmatis. Os resultados obtidos utilizando cepas de referência demonstraram que a PCR multiplex pode ser uma ferramenta rápida, sensível e específica na diferenciação de micobactérias. This study aimed to optimize a method based on the polymerase chain reaction - multiplex PCR - for differentiation of mycobacteria species of interest for public health. The multiplex PCR was based on simultaneous amplification of the hsp65 gene, which is present in all species of the Mycobacterium genus, the dnaJ gene, which is present only in Mycobacterium tuberculosis and Mycobacterium avium and the IS6110 insertion sequence, which is present in the Mycobacterium tuberculosis complex, generating amplicons of 165 bp, 365 bp and 541 bp, respectively. The detection limit was 1 fg for the hsp65 target, 100 pg for dnaJ and 0.1 fg for IS6110. The multiplex PCR detected down to 100 pg of DNA of Mycobacterium tuberculosis. The system was shown to be specific and sensitive for detection of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. The results obtained using reference strains of mycobacteria showed that multiplex PCR may be a fast, sensitive and specific tool for differentiation of ...