Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting
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-HR...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Fundação Oswaldo Cruz (FIOCRUZ)
2013-02-01
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| Series: | Memorias do Instituto Oswaldo Cruz |
| Subjects: | |
| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000100017&lng=en&tlng=en |
| _version_ | 1797713370428735488 |
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| author | Joas Lucas da Silva Gabriela Guimaraes Sousa Leite Gisele Medeiros Bastos Beatriz Cacciacarro Lucas Daniel Keniti Shinohara Joice Sayuri Takinami Marcelo Miyata Cristina Moreno Fajardo André Ducati Luchessi Clarice Queico Fujimura Leite Rosilene Fressatti Cardoso Rosario Dominguez Crespo Hirata Mario Hiroyuki Hirata |
| author_facet | Joas Lucas da Silva Gabriela Guimaraes Sousa Leite Gisele Medeiros Bastos Beatriz Cacciacarro Lucas Daniel Keniti Shinohara Joice Sayuri Takinami Marcelo Miyata Cristina Moreno Fajardo André Ducati Luchessi Clarice Queico Fujimura Leite Rosilene Fressatti Cardoso Rosario Dominguez Crespo Hirata Mario Hiroyuki Hirata |
| author_sort | Joas Lucas da Silva |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-12T07:35:30Z |
| format | Article |
| id | doaj.art-bd0e8ccbfc4d4f67b32fbfbc0d315f44 |
| institution | Directory Open Access Journal |
| issn | 1678-8060 |
| language | English |
| last_indexed | 2024-03-12T07:35:30Z |
| publishDate | 2013-02-01 |
| publisher | Fundação Oswaldo Cruz (FIOCRUZ) |
| record_format | Article |
| series | Memorias do Instituto Oswaldo Cruz |
| spelling | doaj.art-bd0e8ccbfc4d4f67b32fbfbc0d315f442023-09-02T21:33:10ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz1678-80602013-02-01108110610910.1590/S0074-02762013000100017S0074-02762013000100017Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution meltingJoas Lucas da Silva0Gabriela Guimaraes Sousa Leite1Gisele Medeiros Bastos2Beatriz Cacciacarro Lucas3Daniel Keniti Shinohara4Joice Sayuri Takinami5Marcelo Miyata6Cristina Moreno Fajardo7André Ducati Luchessi8Clarice Queico Fujimura Leite9Rosilene Fressatti Cardoso10Rosario Dominguez Crespo Hirata11Mario Hiroyuki Hirata12Universidade de São PauloUniversidade de São PauloUniversidade de São PauloUniversidade de São PauloUniversidade de São PauloUniversidade de São PauloUniversidade de São PauloUniversidade de São PauloUniversidade de São PauloUniversidade Estadual PaulistaUniversidade Estadual de MaringáUniversidade de São PauloUniversidade de São PauloQuantitative 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.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000100017&lng=en&tlng=endrug resistancerifampicinMycobacterium tuberculosis |
| spellingShingle | Joas Lucas da Silva Gabriela Guimaraes Sousa Leite Gisele Medeiros Bastos Beatriz Cacciacarro Lucas Daniel Keniti Shinohara Joice Sayuri Takinami Marcelo Miyata Cristina Moreno Fajardo André Ducati Luchessi Clarice Queico Fujimura Leite Rosilene Fressatti Cardoso Rosario Dominguez Crespo Hirata Mario Hiroyuki Hirata Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting Memorias do Instituto Oswaldo Cruz drug resistance rifampicin Mycobacterium tuberculosis |
| title | 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_short | 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 |
| topic | drug resistance rifampicin Mycobacterium tuberculosis |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000100017&lng=en&tlng=en |
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