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

Memórias do Instituto Oswaldo Cruz

Joas Lucas Da Silva¹  Gabriela Guimaraes Sousa Leite¹  Gisele Medeiros Bastos¹  Beatriz Cacciacarro Lucas¹  Daniel Keniti Shinohara¹  Joice Sayuri Takinami¹  Marcelo Miyata¹  Cristina Moreno Fajardo¹  André Ducati Luchessi¹  Clarice Queico Fujimura Leite²  Rosilene Fressatti Cardoso²  Rosario Dominguez Crespo Hirata¹  Mario Hiroyuki Hirata¹ 

[1] ,Universidade de São Paulo Faculdade de Ciências Farmacêuticas São Paulo SP ,Brasil

关键词: drug resistance;    rifampicin;    Mycobacterium tuberculosis;   

DOI  :  10.1590/S0074-02762013000100017

来源: SciELO

【 摘 要 】

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.

【 授权许可】

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