Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

• Autor(a) principal: Silva,Joas Lucas da
• Data de Publicação: 2013
• Outros Autores: 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
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Memórias do Instituto Oswaldo Cruz
• Texto Completo: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000100017
• topic: drug resistance; rifampicin; Mycobacterium tuberculosis

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.