Memorias do Instituto Oswaldo Cruz (Feb 2013)

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

  • 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

Journal volume & issue
Vol. 108, no. 1
pp. 106 – 109

Abstract

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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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