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.
Highlights
Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains as a major public health problem
We have developed a molecular method for detection of RIF resistance in M. tuberculosis by padlocks probes, rolling circle amplification (RCA) and a magnetic nanobead-based readout; volume amplified magnetic nanobead detection assay (VAM-NDA)
We have developed a molecular method for detection of RIF resistance in M. tuberculosis by padlock probes and a magnetic bead-based readout (Figure 1)
Summary
Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains as a major public health problem. Increasing resistance to anti-TB drugs severely threatens the control of the disease. Prompt detection of drug-resistant M. tuberculosis strains is crucial for prescription of appropriate treatment. In order to quickly detect drug-resistant M. tuberculosis it is essential to use molecular based diagnostic methods, which can be performed within a day. Chromosomal mutations are the genetic basis for drug resistance in M. tuberculosis [1,2]. The effective first line anti-TB drug rifampicin (RIF) inhibits transcription by binding to the ßsubunit (encoded by rpoB) of the RNA polymerase [3]. Resistance to RIF in M. tuberculosis is almost entirely associated with mutations within an 81-bp region of the rpoB gene, called the RIF resistancedetermining region (RRDR), comprising codons 507 to 533 [2,4]. A substitution in the first or second nucleotide position of codons
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