Detection of isoniazid-resistant Mycobacterium tuberculosis strains by a multiplex allele-specific PCR assay targeting katG codon 315 variation.
We describe a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene codon 315, including AGC-->ACC and ACA (Ser-->Thr) substitutions that confer resistance to isoniazid (INH) in Mycobacterium tuberculosis clinical isolates. The 315 ACC allele is found in the majority of Inh(r) strains worldwide, especially in areas with a high incidence of tuberculosis. The 315 ACA allele is characteristic of the New York City multidrug-resistant (MDR) strain W and its progenies in the United States. The mutations in katG315 are revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. Initially optimized on the purified DNA samples, the assay was then tested on crude cell lysates and auramine-stained sputum slide preparations with the same reproducibility and interpretability of profiles generated by agarose gel electrophoresis. The MAS-PCR assay can be used for the detection of resistance to INH in clinical laboratories in regions with a high prevalence of MDR M. tuberculosis strains.
Mokrousov
Igor
I
Laboratory of Molecular Microbiology, St. Petersburg Pasteur Institute, 197101 St. Petersburg, Russia. miv@IM4520.spb.edu
Otten
Tatiana
T
Filipenko
Maxim
M
Vyazovaya
Anna
A
Chrapov
Eugeny
E
Limeschenko
Elena
E
Steklova
Lidia
L
Vyshnevskiy
Boris
B
Narvskaya
Olga
O
D000293:Adolescent; D000328:Adult; D000483:Alleles; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D014644:Genetic Variation; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D010544:Peroxidases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012426:Russia; D014397:Tuberculosis, Pulmonary / Q000382:microbiology
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003062:Codon; D004269:DNA, Bacterial; D010544:Peroxidases; C076136:catalase HPI; D007538:Isoniazid