Author(s):
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 [UNESP] ; Cardoso, Rosilene Fressatti ; Hirata, Rosario Dominguez Crespo ; Hirata, Mario Hiroyuki
Date: 2014
Persistent ID: http://hdl.handle.net/11449/7948
Origin: Oasisbr
Subject(s): drug resistance; rifampicin; Mycobacterium tuberculosis
Description
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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.
Universidade de São Paulo Faculdade de Ciências Farmacêuticas
Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas
Universidade Estadual de Maringá (UEM) Departamento de Análises Clínicas
Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas
