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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060
EISSN: 1678-8060
Vol. 108, No. 1, 2013, pp. 106-109
Bioline Code: oc13017
Full paper language: English
Document type: Research Article
Document available free of charge

Memórias do Instituto Oswaldo Cruz, Vol. 108, No. 1, 2013, pp. 106-109

 en Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis check for this species in other resources by quantitative polymerase chain reaction-high-resolution melting
da Silva, Joas Lucas; 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

Abstract

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis check for this species in other resources . 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 check for this species in other resources and can be extracted in large amounts.

Keywords
drug resistance - rifampicin - Mycobacterium tuberculosis

 
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