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
Article
em En
| LILACS
| ID: lil-666052
Biblioteca responsável:
BR1.1
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.
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Texto completo:
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Base de dados:
LILACS
Assunto principal:
Rifampina
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Proteínas de Bactérias
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Farmacorresistência Bacteriana
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Antibióticos Antituberculose
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Mutação
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Mycobacterium tuberculosis
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article