title: Detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis using denaturing gradient gel electrophoresis.
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.
authors.affiliation: Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, STCBM Building, 15355 Lambda Drive, San Antonio, TX 78245, USA. mccammon@uthscsa.edu
authors.forename: Mark T
authors.initials: MT
authors.lastname: McCammon
authors.forename: John S
authors.initials: JS
authors.lastname: Gillette
authors.forename: Derek P
authors.initials: DP
authors.lastname: Thomas
authors.forename: Srinivas V
authors.initials: SV
authors.lastname: Ramaswamy
authors.forename: Edward A
authors.initials: EA
authors.lastname: Graviss
authors.forename: Barry N
authors.initials: BN
authors.lastname: Kreiswirth
authors.forename: Jan
authors.initials: J
authors.lastname: Vijg
authors.forename: Teresa N
authors.initials: TN
authors.lastname: Quitugua
mesh_terms: D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001483:Base Sequence; D003062:Codon; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D012293:Rifampin / Q000494:pharmacology*; D017422:Sequence Analysis, DNA
chemical_list: D000904:Antibiotics, Antitubercular; D003062:Codon; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin