Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Outros Autores: | , , , , , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1590/S0074-02762013000100017 http://hdl.handle.net/11449/7948 |
Resumo: | 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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Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution meltingdrug resistancerifampicinMycobacterium tuberculosisQuantitative 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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade de São Paulo Faculdade de Ciências FarmacêuticasUniversidade Estadual Paulista Faculdade de Ciências FarmacêuticasUniversidade Estadual de Maringá (UEM) Departamento de Análises ClínicasUniversidade Estadual Paulista Faculdade de Ciências FarmacêuticasInstituto Oswaldo Cruz, Ministério da SaúdeUniversidade de São Paulo (USP)Universidade Estadual Paulista (Unesp)Universidade Estadual de Maringá (UEM)Silva, Joas Lucas daLeite, Gabriela Guimaraes SousaBastos, Gisele MedeirosLucas, Beatriz CacciacarroShinohara, Daniel KenitiTakinami, Joice SayuriMiyata, MarceloFajardo, Cristina MorenoLuchessi, André DucatiLeite, Clarice Queico Fujimura [UNESP]Cardoso, Rosilene FressattiHirata, Rosario Dominguez CrespoHirata, Mario Hiroyuki2014-05-20T13:25:07Z2014-05-20T13:25:07Z2013-02-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article106-109application/pdfhttp://dx.doi.org/10.1590/S0074-02762013000100017Memórias do Instituto Oswaldo Cruz. Instituto Oswaldo Cruz, Ministério da Saúde, v. 108, n. 1, p. 106-109, 2013.0074-0276http://hdl.handle.net/11449/794810.1590/S0074-02762013000100017S0074-02762013000100017WOS:000315335800017S0074-02762013000100017.pdf2114570774349859SciELOreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengMemórias do Instituto Oswaldo Cruz2.8331,172info:eu-repo/semantics/openAccess2023-12-08T06:25:37Zoai:repositorio.unesp.br:11449/7948Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462023-12-08T06:25:37Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting |
title |
Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting |
spellingShingle |
Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting Silva, Joas Lucas da drug resistance rifampicin Mycobacterium tuberculosis |
title_short |
Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting |
title_full |
Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting |
title_fullStr |
Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting |
title_full_unstemmed |
Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting |
title_sort |
Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting |
author |
Silva, Joas Lucas da |
author_facet |
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 |
author_role |
author |
author2 |
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 |
author2_role |
author author author author author author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade de São Paulo (USP) Universidade Estadual Paulista (Unesp) Universidade Estadual de Maringá (UEM) |
dc.contributor.author.fl_str_mv |
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 |
dc.subject.por.fl_str_mv |
drug resistance rifampicin Mycobacterium tuberculosis |
topic |
drug resistance rifampicin Mycobacterium tuberculosis |
description |
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. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013-02-01 2014-05-20T13:25:07Z 2014-05-20T13:25:07Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1590/S0074-02762013000100017 Memórias do Instituto Oswaldo Cruz. Instituto Oswaldo Cruz, Ministério da Saúde, v. 108, n. 1, p. 106-109, 2013. 0074-0276 http://hdl.handle.net/11449/7948 10.1590/S0074-02762013000100017 S0074-02762013000100017 WOS:000315335800017 S0074-02762013000100017.pdf 2114570774349859 |
url |
http://dx.doi.org/10.1590/S0074-02762013000100017 http://hdl.handle.net/11449/7948 |
identifier_str_mv |
Memórias do Instituto Oswaldo Cruz. Instituto Oswaldo Cruz, Ministério da Saúde, v. 108, n. 1, p. 106-109, 2013. 0074-0276 10.1590/S0074-02762013000100017 S0074-02762013000100017 WOS:000315335800017 S0074-02762013000100017.pdf 2114570774349859 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Memórias do Instituto Oswaldo Cruz 2.833 1,172 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
106-109 application/pdf |
dc.publisher.none.fl_str_mv |
Instituto Oswaldo Cruz, Ministério da Saúde |
publisher.none.fl_str_mv |
Instituto Oswaldo Cruz, Ministério da Saúde |
dc.source.none.fl_str_mv |
SciELO reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
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1797789943988224000 |