Rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in Mycobacterium tuberculosis clinical isolates using non-fluorescent low-density DNA microarrays

Background: A new, fast ‘low cost and density’ DNA microarray (LCD array), designed for the detection of mutations that confer isoniazid or rifampicin resistance in Mycobacterium tuberculosis isolates, has been developed and was evaluated using 46 resistant clinical isolates from Barcelona. Methods:...

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Bibliographic Details
Published in:Journal of antimicrobial chemotherapy 2006-05, Vol.57 (5), p.825-831
Main Authors: Aragón, Lina Marcela, Navarro, Ferran, Heiser, Volker, Garrigó, Montserrat, Español, Montserrat, Coll, Pere
Format: Article
Language:English
Subjects:
Antibiotics. Antiinfectious agents. Antiparasitic agents
Antitubercular Agents - pharmacology
Bacterial diseases
Bacterial Proteins - genetics
Biological and medical sciences
Catalase - genetics
DNA, Bacterial - genetics
DNA-Directed RNA Polymerases
drug resistance
Drug Resistance, Multiple, Bacterial - genetics
Genes, Bacterial
Human bacterial diseases
Humans
Infectious diseases
Isoniazid - pharmacology
katG
mabA-inhA
Medical sciences
Mutation
Mycobacterium tuberculosis
Mycobacterium tuberculosis - drug effects
Mycobacterium tuberculosis - genetics
Mycobacterium tuberculosis - isolation & purification
Oligonucleotide Array Sequence Analysis - methods
Oxidoreductases - genetics
Pharmacology. Drug treatments
Promoter Regions, Genetic
Regulatory Sequences, Nucleic Acid
Rifampin - pharmacology
rpoB
tuberculosis
Tuberculosis - drug therapy
Tuberculosis - microbiology
Tuberculosis and atypical mycobacterial infections
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Summary:Background: A new, fast ‘low cost and density’ DNA microarray (LCD array), designed for the detection of mutations that confer isoniazid or rifampicin resistance in Mycobacterium tuberculosis isolates, has been developed and was evaluated using 46 resistant clinical isolates from Barcelona. Methods: LCD chips are pre-structured polymer supports using a non-fluorescent detection principle based on the precipitation of a clearly visible dark substrate. One LCD chip consists of eight identical microarrays, designed to detect mutations within the 90 bp rpoB region, codon 315 in the katG gene and the mabA-inhA regulatory region. A total of 22 strains with a katG 315 mutation, 19 strains with alterations in the mabA-inhA regulatory region and 16 strains with mutations in the rpoB region, characterized previously, were studied. Results: The identification of S315T and S315N mutations using the LCD was 100% concordant with the sequencing data. A strain with the S315R mutation, which is not tiled on the LCD array, was detected by the absence of hybridization using the wild-type probe. Of 19 strains with low-level isoniazid resistance related to the mabA-inhA regulatory region, 18 were identified correctly. The detection of mutations in the rpoB region was 93.8% concordant with the sequencing data. One mabA-inhA and rpoB mutated strain showed a cross-hybridization. Conclusions: The LCD array protocol takes 45 min (15 min ‘hands-on’ time) after prior PCR amplification. Only minimal laboratory equipment is required. LCD arrays provide a rapid and economical method to characterize mutations in codon 315 of the katG gene, in the mabA-inhA regulatory region and in the rpoB gene.
ISSN:0305-7453
1460-2091
DOI:10.1093/jac/dkl058