Deteção de Mycobacterium tuberculosis em amostras clínicas por reação em cadeia da polimerase utilizando primers baseados na região intergênica plcB-plcC / Using polymerase chain reaction with primers based on the plcB-plcC intergenic region to detect Mycobacterium tuberculosis in clinical samples

OBJECTIVE: To develop a system for the molecular diagnosis of tuberculosis by polymerase chain reaction (PCR), constructing primers based on the difference in gene organization of the intergenic region of phospholipase C (plcB-plcC region), which differentiates Mycobacterium tuberculosis from other mycobacteria. METHODS: A PCR product of the expected size (432 bp) was obtained from M. tuberculosis and M. africanum only. A total of 33 mycobacterial isolates and 273 clinical samples from patients suspected of having tuberculosis were examined. These were used in the comparative study of the PCR technique versus culture. RESULTS: For PCR versus culture, the data showed 93.8 percent accuracy (p < 0.0001), 93.1 percent sensitivity (CI: 88.7-96.0), and 96.4 percent specificity (CI: 96.1-99.4). The Kappa value (0.82) shows that there was a near-perfect concordance between the two tests. CONCLUSION: The use of the plcB-plcC region in PCR amplification was found to be an important and reliable tool for the specific diagnosis of tuberculosis in the samples analyzed.

OBJETIVO: Desenvolver um sistema para o diagnóstico molecular da tuberculose por reação em cadeia da polimerase, do inglês polymerase chain reaction (PCR), pela construção de primers baseados na diferença da organização de uma região intergênica da fosfolipase (phospholipase) C (região plcB-plcC), que diferencia Mycobacterium tuberculosis das outras micobactérias. MÉTODOS: Um produto de PCR com o tamanho esperado (432 pb) foi obtido somente de M. tuberculosis e M. africanum. Um total de 33 isolados micobacterianos e 273 amostras clínicas de pacientes com suspeita de tuberculose foram examinados. Estes foram submetidos ao estudo comparativo da técnica de PCR contra o cultivo. RESULTADOS: Os dados mostraram 93,8 por cento de exatidão para PCR contra o cultivo (p < 0,0001), 93,1 por cento de sensibilidade (IC: 88,7-96,0) e especificidade de 96,4 por cento (IC: 96,1-99,4). O valor de Kappa foi de 0,82, demonstrando um alinhamento perfeito para a verificação do grau de concordância entre os testes. CONCLUSÃO: O uso da região plcB-plcC para a amplificação por PCR é mostrado como uma ferramenta importante e de confiança para o diagnóstico específico de tuberculose nas amostras clínicas analisadas.

Superoxide dismutases in the entomopathogenic fungus Metarhizium anisopliae

Control of gene expression is a key subject in Molecular Biology. Superoxide dismutases are essential enzymes to protect living organisms against toxicity of radicals generated by the metabolism and represent an ideal system to study gene regulation. Filamentous fungi are extensively used as model eukaryotic systems and some representatives are important microorganisms in the biological control of insects in agriculture. Metarhizium anisopliae is employed at a commercial scale to control insects in sugar-cane plantations and pastures in Brazil and is currently the best studied entomopathogenic fungus. It possesses three SOD activities, CuZnSOD, MnSOD and Fe SOD. The iron enzyme is found in fungi for the first time. A gene coding for SOD was cloned by PCR amplification, partially sequenced and is under characterization. Transformation systems are developed but rendering poor efficiencies. Homologous genes have been isolated and should increase transformation yields.