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

• Main Authors: Joas Lucas da Silva, Gabriela Guimaraes Sousa Leite, Gisele Medeiros Bastos, Beatriz Cacciacarro Lucas, Daniel Keniti Shinohara, Joice Sayuri Takinami, Marcelo Miyata, Cristina Moreno Fajardo, André Ducati Luchessi, Clarice Queico Fujimura Leite, Rosilene Fressatti Cardoso, Rosario Dominguez Crespo Hirata, Mario Hiroyuki Hirata
• Format: Article
• Language: English
• Published: Instituto Oswaldo Cruz, Ministério da Saúde 2013-02-01
• Series: Memórias do Instituto Oswaldo Cruz.
• Subjects: drug resistance; rifampicin; Mycobacterium tuberculosis
• Online Access: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000100017
• ISSN: 0074-0276; 1678-8060

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.