Detection of ethambutol-resistant Mycobacterium tuberculosis strains by multiplex allele-specific PCR assay targeting embB306 mutations.
We describe a multiplex allele-specific (MAS)-PCR assay to detect simultaneously mutations in the first and third bases of the embB gene codon 306ATG. These mutations are known to confer ethambutol (EMB) resistance in the majority of clinical Mycobacterium tuberculosis isolates worldwide. The mutated bases are revealed depending on the presence or absence of the respective indicative fragments amplified from the embB306 wild-type allele. Initially optimized on purified DNA samples, the assay was tested on crude cell lysates and auramine-stained sputum slide DNA preparations with the same reproducibility and interpretability of the generated profiles in agarose gel electrophoresis. Since EMB resistance is generally linked to multiple-drug resistance (MDR), the MAS-PCR assay for EMB resistance detection can be used in clinical laboratory practice in areas with a high prevalence and a high transmission rate of MDR-EMB-resistant tuberculosis.
Mokrousov
Igor
I
Laboratory of Molecular Microbiology, Pasteur Institute, 14, Mira Street, St. Petersburg 197101, Russia. miv@IM4520.spb.edu
Narvskaya
Olga
O
Limeschenko
Elena
E
Otten
Tatiana
T
Vyshnevskiy
Boris
B
D000483:Alleles; D000995:Antitubercular Agents / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers; D004352:Drug Resistance, Microbial; D004977:Ethambutol / Q000494:pharmacology*; D006801:Humans; D016296:Mutagenesis; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D010430:Pentosyltransferases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping
D000995:Antitubercular Agents; D017931:DNA Primers; D004977:Ethambutol; D010430:Pentosyltransferases; C101967:arabinosyltransferase