List of oligonucleotides used in this assay.

a<p>A + sign before nt indicate the LNA residues. As implicated in PLP_A: the bold font indicated the Rotavirus targeting padlock probe arms, Italic font indicates the AluI restriction site, and the underlined sequence the sequence for detection probe hybridization.</p><p>List of oligonucleotides used in this assay.</p

Analysis of patient samples.

<p>cDNA was prepared from 22 patient samples collected at Uppsala University hospital. (A) PCR positive samples. (B) PCR negative samples. Plotted is the number of rolling circle product - (negative+3 SD) on the y-axis and patient samples on the x-axis. Error bars ±1 s.d.; n = 2.</p

Schematic summary of assay principle.

<p>(1) RNA is extracted from patient samples and (2) cDNA is synthesized using biotinylated primers. (3) The DNA is denatured and (4) padlock probes are hybridized and ligated to their complementary target sequence. (5) This complex is captured onto magnetic beads to wash away unbound probes and (6) amplified by rolling circle amplification (RCA) for 20 min. (7) The rolling circle products (RCPs) are monomerized and (8) a second round of RCA is performed. (9) Fluorescently labeled oligonucleotides are hybridized to the RCPs for detection in a microfluidic setup using a confocal microscope.</p

Analytical sensitivity of the method.

<p>Short synthetic 5′ biotinylated DNA templates are serially diluted to assess the analytical sensitivity of our assay. The y-axis shows the number of rolling circle products (RCPs) and the x-axis the copy number of a synthetic biotinylated DNA target. The negative sample is a no template control. Error bars ±1 s.d.; n = 3.</p

Padlock probe design.

<p>(A) The sequence of the six padlock probes with the haplotyped degenerations and ligation site. (B) Alignment of 214 Human Rotaviruses. The variations in Rotavirus were mapped using BLASTn and ConSort. The length of the black bars represents the frequency of variation as an average percentage conservation at each nt position (y-axis).</p

Specificity testing of wild type probe system.

<p>The system was tested on the reference strain <i>M. tuberculosis</i> H37Rv (representing wild type <i>rpoB</i>), eight <i>M. tuberculosis</i> clinical isolates, each harboring an rpoB 516 TAC, 516 GTC, 526 CGC, 526 CTC, 526 TAC, 526 AAC, 531 TTG or 531 TGG mutation, and a synthetic target representing an <i>rpoB</i> 526 GAC mutation. RCA products were hybridized to fluorescent dye-coupled oligonucleotide and visualized in a confocal microscope. NC: negative control.</p

Complete assay tested on <i>M. tuberculosis</i> DNA samples.

<p>Complete assay (wild type probe system, cocktail of nine mutant-specific padlock probes, <i>M. tuberculosis</i> complex (MTC) padlock probe, and negative controls for respective probe or probe mix (NC)) was evaluated on four DNA samples from the RIF-susceptible reference strain <i>M. tuberculosis</i> H37Rv (wild type <i>rpoB</i>) and three RIF-resistant <i>M. tuberculosis</i> clinical isolates harboring a 516 GTC, 526 TAC or a 531 TTG <i>rpoB</i> mutation by the magnetic nanobead-based readout. To account for differences in iron-oxide content between samples, the data were normalized using the constant value of the in-phase component of the volume susceptibility, .</p

Detection of Rifampicin Resistance in <i>Mycobacterium tuberculosis</i> by Padlock Probes and Magnetic Nanobead-Based Readout

<div><p>Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant <i>Mycobacterium tuberculosis</i> 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 <i>M. tuberculosis</i> based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in <i>M. tuberculosis</i>, i.e. at codons 516, 526 and 531 in the gene <i>rpoB</i>. 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 <i>M. tuberculosis</i> 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.</p></div

Specificity testing of the <i>M. tuberculosis</i> complex padlock probe.

<p>The probe, designed to target the 16S–23S intergenic spacer region, was tested on five <i>M. tuberculosis</i> complex species; <i>M. africanum</i>, <i>M. bovis</i>, <i>M. canetti</i>, <i>M. microti</i> and <i>M. tuberculosis</i>, and on five nontuberculous mycobacterial species; <i>M. avium</i>, <i>M. interjectum</i>, <i>M. kansasaii</i>, <i>M. marinum</i>, <i>M. szulgai</i>. RCA products were hybridized to fluorescent dye-coupled oligonucleotide and visualized in a confocal microscope. NC: negative control.</p

Specificity and multiplexability testing of the mutant-specific padlock probes.

<p>The probes were tested in single-, du-, or tri-plex and nine-plex (all mutant-specific padlock probes) on eight <i>M. tuberculosis</i> clinical isolates harboring an <i>rpoB</i> 516 TAC or GTC mutation (A), an <i>rpoB</i> 526 CGC or 526 CTC mutation (B), an <i>rpoB</i> 526 AAC or 526 TAC mutation (C), an <i>rpoB</i> 531 TTG or 531 TGG mutation (D), on a synthetic target with an <i>rpoB</i> 526 GAC mutation (C), and on the reference strain <i>M. tuberculosis</i> H37Rv (representing wild type <i>rpoB</i>) (A–D). RCA products were hybridized to fluorescent dye-coupled oligonucleotide and visualized in a confocal microscope. NC: negative control.</p