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

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dc.contributor.authorSilva, Joas Lucas da
dc.contributor.authorLeite, Gabriela Guimaraes Sousa
dc.contributor.authorBastos, Gisele Medeiros
dc.contributor.authorLucas, Beatriz Cacciacarro
dc.contributor.authorShinohara, Daniel Keniti
dc.contributor.authorTakinami, Joice Sayuri
dc.contributor.authorMiyata, Marcelo
dc.contributor.authorFajardo, Cristina Moreno
dc.contributor.authorLuchessi, André Ducati
dc.contributor.authorLeite, Clarice Queico Fujimura [UNESP]
dc.contributor.authorCardoso, Rosilene Fressatti
dc.contributor.authorHirata, Rosario Dominguez Crespo
dc.contributor.authorHirata, Mario Hiroyuki
dc.contributor.institutionUniversidade de São Paulo (USP)
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.contributor.institutionUniversidade Estadual de Maringá (UEM)
dc.date.accessioned2014-05-20T13:25:07Z
dc.date.available2014-05-20T13:25:07Z
dc.date.issued2013-02-01
dc.description.abstractQuantitative 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.en
dc.description.affiliationUniversidade de São Paulo Faculdade de Ciências Farmacêuticas
dc.description.affiliationUniversidade Estadual Paulista Faculdade de Ciências Farmacêuticas
dc.description.affiliationUniversidade Estadual de Maringá (UEM) Departamento de Análises Clínicas
dc.description.affiliationUnespUniversidade Estadual Paulista Faculdade de Ciências Farmacêuticas
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.description.sponsorshipConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.format.extent106-109
dc.identifierhttp://dx.doi.org/10.1590/S0074-02762013000100017
dc.identifier.citationMemórias do Instituto Oswaldo Cruz. Instituto Oswaldo Cruz, Ministério da Saúde, v. 108, n. 1, p. 106-109, 2013.
dc.identifier.doi10.1590/S0074-02762013000100017
dc.identifier.fileS0074-02762013000100017.pdf
dc.identifier.issn0074-0276
dc.identifier.lattes2114570774349859
dc.identifier.scieloS0074-02762013000100017
dc.identifier.urihttp://hdl.handle.net/11449/7948
dc.identifier.wosWOS:000315335800017
dc.language.isoeng
dc.publisherInstituto Oswaldo Cruz, Ministério da Saúde
dc.relation.ispartofMemórias do Instituto Oswaldo Cruz
dc.relation.ispartofjcr2.833
dc.relation.ispartofsjr1,172
dc.rights.accessRightsAcesso aberto
dc.sourceSciELO
dc.subjectdrug resistanceen
dc.subjectrifampicinen
dc.subjectMycobacterium tuberculosisen
dc.titlePlasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution meltingen
dc.typeArtigo
unesp.author.lattes2114570774349859
unesp.author.orcid0000-0002-2758-8102[9]
unesp.campusUniversidade Estadual Paulista (Unesp), Faculdade de Ciências Farmacêuticas, Araraquarapt

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Artigos - Ciências Biológicas - FCFAR