title: Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR.
abstract: Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.
authors.affiliation: Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences, Osaka, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Wada
authors.forename: Shinji
authors.initials: S
authors.lastname: Maeda
authors.forename: Aki
authors.initials: A
authors.lastname: Tamaru
authors.forename: Shigeyoshi
authors.initials: S
authors.lastname: Imai
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hase
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kobayashi
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes; D024881:Drug Resistance, Bacterial / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D015342:DNA Probes