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
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.
Servei de Microbiología, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain.
Lina Marcela
LM
Aragón
Ferran
F
Navarro
Volker
V
Heiser
Montserrat
M
Garrigó
Montserrat
M
Español
Pere
P
Coll
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D002374:Catalase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010088:Oxidoreductases / Q000235:genetics; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012293:Rifampin / Q000494:pharmacology; D014376:Tuberculosis / Q000188:drug therapy / Q000382:microbiology
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; D012321:DNA-Directed RNA Polymerases; D007538:Isoniazid; D012293:Rifampin