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.