Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication.
Graziele Lima Bello, Franciele Costa Leite Morais, Jonas Michel Wolf, Mirela Gehlen, Tainá Dos Santos Soares, Maria Laura Halon, Regina Bones Barcellos, Maria Lucia Rosa Rossetti
Author Information
Graziele Lima Bello: Universidade Luterana do Brasil (ULBRA), Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Canoas, RS, Brasil.
Franciele Costa Leite Morais: Universidade Luterana do Brasil (ULBRA), Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Canoas, RS, Brasil.
Jonas Michel Wolf: Universidade Luterana do Brasil (ULBRA), Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Canoas, RS, Brasil.
Mirela Gehlen: Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pós-Graduação em Pneumologia, Porto Alegre, RS, Brasil.
Tainá Dos Santos Soares: Universidade Luterana do Brasil (ULBRA), Curso de Biomedicina, Canoas, RS, Brasil. Electronic address: taina.soares@rede.ulbra.br.
Maria Laura Halon: Secretaria do Estado do Rio Grande do Sul (SES/ RS), Centro de Desenvolvimento Científico e Tecnológico (CDCT), Porto Alegre, RS, Brasil.
Regina Bones Barcellos: Secretaria do Estado do Rio Grande do Sul (SES/ RS), Centro de Desenvolvimento Científico e Tecnológico (CDCT), Porto Alegre, RS, Brasil; Universidade Federal do Rio de Janeiro (UFRJ), Programa de Pós-Graduação em Clínica Médica, Rio de Janeiro, RJ, Brasil.
Maria Lucia Rosa Rossetti: Universidade Luterana do Brasil (ULBRA), Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Canoas, RS, Brasil; Universidade Federal do Rio de Janeiro (UFRJ), Programa de Pós-Graduação em Clínica Médica, Rio de Janeiro, RJ, Brasil. Electronic address: maria.rossetti@ulbra.br.
Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14 CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.
