Detection of rpoB Gene Mutation in Rifampin-Resistant M. Tuberculosis by Oligonucleotide Chip

Park, Soonkew; Lee, Minki; Chung, Byungseon; Kim, Cheolmin; Chang, Chulhun; Park, Heekyung; Jang, Hyunjung; Park, Seungkyu; Song, Sundae

Abstract

BACKGROUND
Oligonucleotide chip technology has proven to be a very useful tool in the rapid diagnosis of infectious disease. Rifampin resistance is considered as a useful marker of multidrug-resistance in tuberculosis. Mutations in the rpoB gene coding Î² subunit of RNA polymerase represent the main mechanism of rifampin resistance. The purpose of this study was to develop a diagnosis kit using oligonucleotide chip for the rapid and accurate detection of rifampin-resistance in Mycobacterium tuberculosis. METHOD: Tle sequence specific probes for mutations in the rpoB gene were designed and spotted onto the glass slide, oligonucleotide chip. 38 clinical isolates of Mycobacterium were tested. A part of rpoB was amplified, labelled, and hybridized on the oligonucleotide chip with probes. Results were analyzed with a laser scanner. Direct sequencing was done to verify the results. RESULT: The low-density oligonucleotide chip designed to determine the specific mutations in the rpoB gene of M. tuberculosis accurately detected rifampin resistance associated with mutations in 28 clinical isolates. Mutations at codons 531, 526, and 513 were confirmed by direct sequencing analysis.
CONCLUSION
Mutant detection using oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of multidrug-resistance in M. tuberculosis.