Detection of Point Mutations in the rpoB gene Related to Drug Susceptibility in Mycobacterium Tuberculosis using an Oligonucleotide Chip.
10.4046/trd.2001.50.1.29
- Author:
Hyun Jung KIM
;
Seong keun KIM
;
Tae Sun SHIM
;
Yong Doo PARK
;
Misun PARK
- Publication Type:Original Article
- Keywords:
Mycobacterium Tuberculosis;
rpoB gene;
Oligonucleotide Chip;
Rifampin;
Rifabutin
- MeSH:
DNA;
Drug Therapy;
Glass;
Mass Screening;
Mycobacterium tuberculosis*;
Mycobacterium*;
Point Mutation*;
Rifabutin;
Rifampin;
Tuberculosis
- From:Tuberculosis and Respiratory Diseases
2001;50(1):29-41
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
BACKGROUND: The appearance of multiple-drug-resistant Mycobacterium tuberculosis strains has been seriously compromising successful control of tuberculosis. Rifampin-resistance, caused by mutations in the rpoB gene, can be indicative of multiple-drug-resistance, and its detection is of great importance. The present study aimed to develop an oligonucleotide chip for accurate and convenient screening of drug-resistance. METHODS: In order to detect point mutations in the rpoB gene, an oligonucleotide chip was prepared by immobilizing specific probe DNA to a microscopic slide glass by a chemical reaction. The probe DNA that was selected from the 81 bp core region of the rpoB gene was designed to have mutation sites at the center. A total of 17 mutant probes related to rifampin-resistance including 8 rifabutin-sensitive mutant probes were used in this study. For accurate determination, wild type probes were prepared for each mutation position with an equal length, which enabled a direct comparison of the hybridization intensities between the mutant and wild type. RESULTS: Mycobacterial genomic DNA from clinical samples was tested with the oligonucleotide chip and the results were compared with those of the drug-susceptibility test in addition to sequencing and INNO-LiPA Rif. TB kit test in some cases. Out of 15 samples, the oligonucleotide chip results of 13 samples showed good agreement with the rifabutin-sensitivity results. The two samples with conflicting result also showed a discrepancy between the other tests, suggesting such possibilities as existence of mixed strains and difference in drug-sensitivity. Further verification of these samples in addition to more case studies are required before the final evaluation of the oligonucleotide chip can be made. CONCLUSION: An oligonucleotide chip was developed for the detection of rpoB gene mutations related to drug-susceptibility. The results to date show the potential for using the oligonucleotide chip for accurate and convenient screening of drug-resistance to provide useful information in antituberculosis drug therapy.