Characterization by single strand conformation polymorphism of mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis in strains from Vancouver, Mexico City and New Delhi.
OBJECTIVE
The study was conducted to evaluate usefulness of single strand conformation polymorphism (SSCP) over DNA sequencing in the diagnosis of rifampicin (Rif)-resistant tuberculosis.


METHODS
Forty seven isolates of Mycobacterium tuberculosis (MTB) Rif-resistant and 25 Rif-sensitive were obtained from Vancouver, Mexico city and New Delhi and were analyzed by polymerase chain reaction (PCR) amplification of rpoB gene and the mutations were identified by DNA sequencing and SSCP.


RESULTS
The mutations observed by DNA sequencing in 47 RIF-resistant isolates showed that the most common mutation among Vancouver isolates was in codon 526, Hist-->Arg and in Mexico isolates was in codon 531, Ser-Leu and New Delhi isolates was in codon 516, Asp-->Val. Using fluorescence based PCR-SSCP, it was possible to distinguish Rif-resistant isolates from Rif-sensitive isolates.


CONCLUSIONS
DNA sequencing is a highly accurate method for the detection of mutations associated with drug resistance in tuberculosis but is more expensive and requires special equipment and personnel. SSCP is a simple, accurate method and suitable for analysis of large number of samples and the results are available in less than 72 hours.
Department of Biochemistry, PGIMS, Rohtak.
M
M
Sharma
M
M
Altamirano
H K
HK
Prasad
V P
VP
Myneedo
N
N
Nand
D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases; D006801:Humans; D007194:India; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D012293:Rifampin / Q000009:adverse effects / Q000627:therapeutic use*; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy* / Q000382:microbiology
D010940:Plant Proteins; C101305:RpoB protein, Oryza sativa; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin