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- Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting
- Silva, Joas Lucas da
- Leite, Gabriela Guimaraes Sousa
- Bastos, Gisele Medeiros
- Lucas, Beatriz Cacciacarro
- Shinohara, Daniel Keniti
- Takinami, Joice Sayuri
- Miyata, Marcelo
- Fajardo, Cristina Moreno
- Luchessi, André Ducati
- Leite, Clarice Queico Fujimura
- Cardoso, Rosilene Fressatti
- Hirata, Rosario Dominguez Crespo
- Hirata, Mario Hiroyuki
- Universidade de São Paulo (USP)
- Universidade Estadual Paulista (UNESP)
- Universidade Estadual de Maringá (UEM)
- Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
- Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
- Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.
- Memórias do Instituto Oswaldo Cruz. Instituto Oswaldo Cruz, Ministério da Saúde, v. 108, n. 1, p. 106-109, 2013.
- Instituto Oswaldo Cruz, Ministério da Saúde
- drug resistance
- Mycobacterium tuberculosis
- Acesso aberto
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