title: Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting.
abstract: 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.
authors: Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil. joaslucas@usp.br
authors: Joas Lucas da
authors: JL
authors: Silva
authors: Gabriela Guimaraes Sousa
authors: GG
authors: Leite
authors: Gisele Medeiros
authors: GM
authors: Bastos
authors: Beatriz Cacciacarro
authors: BC
authors: Lucas
authors: Daniel Keniti
authors: DK
authors: Shinohara
authors: Joice Sayuri
authors: JS
authors: Takinami
authors: Marcelo
authors: M
authors: Miyata
authors: Cristina Moreno
authors: CM
authors: Fajardo
authors: André Ducati
authors: AD
authors: Luchessi
authors: Clarice Queico Fujimura
authors: CQ
authors: Leite
authors: Rosilene Fressatti
authors: RF
authors: Cardoso
authors: Rosario Dominguez Crespo
authors: RD
authors: Hirata
authors: Mario Hiroyuki
authors: MH
authors: Hirata
mesh_terms: D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D012293:Rifampin / Q000494:pharmacology*; D017422:Sequence Analysis, DNA
chemical_list: D000904:Antibiotics, Antitubercular; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin