title: Detection of rifampicin resistance in Mycobacterium tuberculosis by padlock probes and magnetic nanobead-based readout.
abstract: Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.
authors: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors: Anna
authors: A
authors: Engström
authors: Teresa
authors: T
authors: Zardán Gómez de la Torre
authors: Maria
authors: M
authors: Strømme
authors: Mats
authors: M
authors: Nilsson
authors: David
authors: D
authors: Herthnek
mesh_terms: D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D008280:Magnetics; D058889:Molecular Typing / Q000379:methods* / Q000592:standards; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D053758:Nanoparticles / Q000737:chemistry*; D012015:Reference Standards; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity
chemical_list: D000904:Antibiotics, Antitubercular; D001426:Bacterial Proteins; D015342:DNA Probes; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin
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