Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting
Mem. Inst. Oswaldo Cruz
;
108(1): 106-109, Feb. 2013. graf, tab
Artigo
em Inglês
| LILACS
| ID: lil-666052
ABSTRACT
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.
Texto completo:
DisponíveL
Índice:
LILACS (Américas)
Assunto principal:
Rifampina
/
Proteínas de Bactérias
/
Farmacorresistência Bacteriana
/
Antibióticos Antituberculose
/
Mutação
/
Mycobacterium tuberculosis
Limite:
Humanos
Idioma:
Inglês
Revista:
Mem. Inst. Oswaldo Cruz
Assunto da revista:
Medicina Tropical
/
Parasitologia
Ano de publicação:
2013
Tipo de documento:
Artigo
País de afiliação:
Brasil
Instituição/País de afiliação:
Universidade de São Paulo/BR
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