Otimização da técnica de MIRU (Mycobacterial Interspersed Repetitive Units) para o estudo epidemiológico de pacientes com tuberculose
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Data
2006-05-29
Autores
Pandolfi, José Rodrigo Cláudio [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O presente trabalho evidencia os esforços realizados na tentativa de se aperfeiçoar a técnica de MIRU (Mycobacterial Interspersed Repetitive Units). Esta utiliza como marcadores, diferenças em fragmentos de DNA não codificadores específicos do Mycobacterium tuberculosis e vem sendo empregada para o estudo epidemiológico da tuberculose, pela facilidade de execução. A técnica de MIRU possibilita uma comparação entre linhagens de diferentes áreas geográficas e permite o rastreamento da movimentação de linhagens individuais. Na otimização os seguintes parâmetros foram determinados: 1. protocolos de purificação de DNA; 2. estratégias de amplificação pela PCR; 3. comparações entre um kit de PCR (PCR Master Mix - PROMEGA) e a utilização da PCR da maneira convencional; 4. testes de variadas condições de amplificação utilizando o kit e os reagentes convencionais de PCR; 5. determinação de protocolos de ciclagem para a amplificação dos fragmentos desejados; 6. teste de amplificação sem a prévia extração de DNA das amostras utilizadas. Após a padronização a metodologia foi utilizada na tipagem de 82 amostras de M. tuberculosis que se encontravam divididas em dois grupos, sendo o primeiro com 49 amostras, provenientes de pacientes do Serviço Especial de Saúde de Araraquara (SESA - USP, Araraquara) e o segundo, composto de 33 amostras de bactérias com perfil de resistência a pirazinamida e a outras drogas, provenientes de Maringá. Nesta etapa foram realizados: 7. PCR para a confirmação de gênero e espécie para M. tuberculosis nas 82 amostras analisadas. 8. Aplicação da técnica de MIRU e análise manual de todas as amostras provenientes de pacientes; 9. Emprego de ferramentas de bioinformática (programa PAST Paleontological Statistics Software Package for Education and Data Analysis) para a análise das amostras...
The present work shows efforts to improve the MIRU (Mycobacterial Interspersed Repetitive Units) assay. This methodology, that has been exploited for fingerprinting the Mycobacterium tuberculosis in molecular epidemiological studies, allows for direct and reliable comparison of results between laboratories and the development of large-scale epidemiological studies. In the optimization of MIRU assay, some parameters were defined: DNA purification protocols and PCR strategies were compared, to select the most practical and economical among them. After the standartization, 82 M. tuberculosis strains, from two different groups were analised. The 49 bacteria strains from the first group were sensible and the 33 from the second one were resistant to pyrazinamide and another drugs as isoniazide and/or rifampicin. To stablish the allelic studies the samples were first confirmed as M. tuberculosis by PCR. Then the MIRU assay was applied and the genetic profile was determined. These results generated dendrograms, obtained by bioinformatic tools. This study showed that any kind of DNA purification and even the use of fresh bacterial culture directly into the PCR tube can be used. When the PCR strategies were compared the utilization of the PCR kit seemed to be more practical and to avoid some mistakes that can happens during the home-made PCR mixture preparation. Furthermore, it allows some dilutions higher than those recommended by the manufacturer. In the same way, the home-made mixture in amounts smaller than those suggested can be used. These experiments indicated that only two annealing temperatures can be used to the PCR with the twelve primer pairs, always in the same MgCl2 concentration, allowing the amplification of more samples at the same time. To generate dendrograms with the allelic data from clinical samples... (Complete abstract click electronic access below)
The present work shows efforts to improve the MIRU (Mycobacterial Interspersed Repetitive Units) assay. This methodology, that has been exploited for fingerprinting the Mycobacterium tuberculosis in molecular epidemiological studies, allows for direct and reliable comparison of results between laboratories and the development of large-scale epidemiological studies. In the optimization of MIRU assay, some parameters were defined: DNA purification protocols and PCR strategies were compared, to select the most practical and economical among them. After the standartization, 82 M. tuberculosis strains, from two different groups were analised. The 49 bacteria strains from the first group were sensible and the 33 from the second one were resistant to pyrazinamide and another drugs as isoniazide and/or rifampicin. To stablish the allelic studies the samples were first confirmed as M. tuberculosis by PCR. Then the MIRU assay was applied and the genetic profile was determined. These results generated dendrograms, obtained by bioinformatic tools. This study showed that any kind of DNA purification and even the use of fresh bacterial culture directly into the PCR tube can be used. When the PCR strategies were compared the utilization of the PCR kit seemed to be more practical and to avoid some mistakes that can happens during the home-made PCR mixture preparation. Furthermore, it allows some dilutions higher than those recommended by the manufacturer. In the same way, the home-made mixture in amounts smaller than those suggested can be used. These experiments indicated that only two annealing temperatures can be used to the PCR with the twelve primer pairs, always in the same MgCl2 concentration, allowing the amplification of more samples at the same time. To generate dendrograms with the allelic data from clinical samples... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Mycobacterium tuberculosis, Epidemiologia molecular, Genotipagem
Como citar
PANDOLFI, José Rodrigo Cláudio. Otimização da técnica de MIRU (Mycobacterial Interspersed Repetitive Units) para o estudo epidemiológico de pacientes com tuberculose. 2006. 82 f. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Química., 2006.