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-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.