title: Simple and rapid discrimination of embB codon 306 mutations in Mycobacterium tuberculosis clinical isolates by a real-time PCR assay using an LNA-TaqMan probe.
abstract: Single nucleotide polymorphisms in the codon 306 of embB gene are most frequently reported in ethambutol-resistant Mycobacterium tuberculosis clinical isolates. Here, we report a simple and rapid real-time PCR assay using a locked nucleic acid (LNA)-TaqMan probe for discriminating the embB306 mutations. The use of a 15-bp chimeric LNA/DNA probe led to a relatively higher level of sensitivity and fluorescence signal in the wild-type embB306 ATG codon. Therefore, the mutant alleles were easily distinguishable from the wild-type allele by their distinctive amplification curve shapes without a melting analysis of the PCR product. This system was fast and less than 0.1 pg of genomic DNA per reaction was needed for detection. Because the results from this real-time assay were absolutely consistent with those from DNA sequencing, it can be effectively applied as a simple and rapid method for primary screening of embB306 mutations that occur frequently in ethambutol-resistant and/or multidrug-resistant M. tuberculosis isolates.
authors.affiliation: Department of Biochemistry, Graduate School of Medicine, Konkuk University, Seoul 140-701, Republic of Korea.
authors.forename: Jee-Hyun
authors.initials: JH
authors.lastname: Yoon
authors.forename: Ji-Sun
authors.initials: JS
authors.lastname: Nam
authors.forename: Kyung-Jin
authors.initials: KJ
authors.lastname: Kim
authors.forename: Young-Tae
authors.initials: YT
authors.lastname: Ro
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D004977:Ethambutol / Q000494:pharmacology*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D015345:Oligonucleotide Probes / Q000235:genetics; D010430:Pentosyltransferases / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D015345:Oligonucleotide Probes; D004977:Ethambutol; C540269:EmbB protein, Mycobacterium tuberculosis; D010430:Pentosyltransferases