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

Silva, Joas Lucas daLeite, Gabriela Guimaraes SousaBastos, Gisele MedeirosLucas, Beatriz CacciacarroShinohara, Daniel KenitiTakinami, Joice SayuriMiyata, MarceloFajardo, Cristina MorenoLuchessi, André DucatiLeite, Clarice Queico Fujimura [UNESP]Cardoso, Rosilene FressattiHirata, Rosario Dominguez CrespoHirata, Mario Hiroyuki2014-05-202014-05-202013-02-01Memórias do Instituto Oswaldo Cruz. Instituto Oswaldo Cruz, Ministério da Saúde, v. 108, n. 1, p. 106-109, 2013.0074-0276http://hdl.handle.net/11449/7948Quantitative 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.106-109engdrug resistancerifampicinMycobacterium tuberculosisPlasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution meltingArtigo10.1590/S0074-02762013000100017S0074-02762013000100017WOS:000315335800017Acesso abertoS0074-02762013000100017.pdf2114570774349859