Detection of rpoB Mutations Associated with Rifampin Resistance in Mycobacterium tuberculosis Using Denaturing Gradient Gel Electrophoresis
Open Access
- 1 June 2005
- journal article
- research article
- Published by American Society for Microbiology in Antimicrobial Agents and Chemotherapy
- Vol. 49 (6), 2200-2209
- https://doi.org/10.1128/aac.49.6.2200-2209.2005
Abstract
Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis . DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of rpoB were developed. These primer sets were used to scan rpoB for DNA alterations in 296 M. tuberculosis patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within rpoB were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable rpoB mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in M. tuberculosis .Keywords
This publication has 22 references indexed in Scilit:
- Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border RegionAntimicrobial Agents and Chemotherapy, 2005
- Genetic Polymorphism inMycobacterium tuberculosisIsolates from Patients with Chronic Multidrug‐Resistant TuberculosisThe Journal of Infectious Diseases, 2004
- Molecular analysis of rifampicin-resistant Mycobacterium tuberculosis isolated in Spain (1996–2001). Description of new mutations in the rpoB gene and review of the literatureInternational Journal of Antimicrobial Agents, 2003
- Use of DNA Extracts from Ziehl-Neelsen-Stained Slides for Molecular Detection of Rifampin Resistance and Spoligotyping of Mycobacterium tuberculosisJournal of Clinical Microbiology, 2003
- Transmission of Drug-Resistant Tuberculosis in Texas and MexicoJournal of Clinical Microbiology, 2002
- Prediction of Drug Resistance in M. tuberculosis: Molecular Mechanisms, Tools, and ApplicationsIUBMB Life, 2002
- rpoB Gene mutations in rifampin-resistant Mycobacterium tuberculosis identified by polymerase chain reaction single-stranded conformational polymorphism.Emerging Infectious Diseases, 2001
- High-Speed, Multicolor Fluorescent Two-Dimensional Gene ScanningGenomics, 2001
- Frequency of rpoB Mutations Inside and Outside the Cluster I Region in Rifampin-Resistant Clinical Mycobacterium tuberculosis IsolatesJournal of Clinical Microbiology, 2001
- Gapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Research, 1997