Summary of the MLPA probes designed and used in this study^a.

a<p>only probes that were functional in this study are shown. Probes are named after the gene and specific codon, nucleotide position (bold), or region they target. Probes are either targeting the mutation (mut) or the wild type (wt) sequence or the presence or absence of an RD. Bacterial DNA sequences are targeted with the left oligo (capital letters), spanning oligo (bold), right oligo (italics), iii =  inosine. xTAG sequences are not shown. RD  =  region of difference.</p

Algorithm applied to all strains analysed for species identification of <i>M. tuberculosis</i> complex and non-tuberculous mycobacteria.

<p>MLPA markers are framed and final NTM species, MTBC lineages or sublineages are shown in bold. The species identification of a sample always starts with the MTBC 16SrRNA marker. As an example the call for the Beijing lineage K1 is highlighted with bold arrows. The following markers are present or absent in a strain belonging to the Beijing K1 lineage: MTBC 16S rRNA (present), TbD1 (present), RD750 (absent), pks15/1–7 (absent), RD105 (present), fbpB-238 (present), muT2-58 (present), acs-1551 (absent), RD131 (present). * as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043240#pone.0043240-Comas2" target="_blank">[25]</a>.</p

Reproducibility of the MLPA assay.

<p>DNA from <i>M. tuberculosis</i> strains 16, 27, 42 and 75 was analysed by MLPA in duplicate and in three experimental replicates. Mean Median Fluorescence Intensity (MFI) values with standard error of the mean (SEM) are shown. Each bar represents one experiment. The dashed line at MFI 150 indicates the set threshold.</p

Overview of the bead-based Multiplex Ligation-dependent Probe Amplification (MLPA) assay.

<p>(a) MLPA oligo design. MLPA oligos were designed to test for (1) single nucleotide polymorphism, the absence (2) or presence (3) of a region of difference (RD), (4) species-specific sequences (b) Hybridisation of MLPA oligos to target DNA. Sequence-specific sequences hybridise to target DNA (DNA1 and DNA2). Each probe consists of a target-specific sequence (grey bars), a unique xTAG (orange bar), forward and reverse primer binding sequences (red and green bars). The MLPA oligos perfectly match to the sequence of DNA1 that harbours a SNP but not to DNA2. (c) Ligation of hybridised oligos. Only oligos that are hybridised directly adjacent to each other are ligated. (d) Amplification of ligated oligos by PCR. All ligated oligos are amplified in a PCR reaction using a single Cy3-labelled forward primer and unlabelled reverse primer. (e) Hybridisation of MLPA products to beads. Amplified probes hybridise to their anti-xTAG coupled to an individual bead species. (f) Analysis of bead-probe complexes on the MAGPIX. A red light emitting diode (LED) and a CCD camera identify first the individual bead species before green LEDs excite the reporter molecules on the probes. The signal is translated into Median Fluorescence Intensity (MFI). For DNA1 a reporter signal is detected on the bead species indicating the presence of the SNP, thus a mutation, in the respective DNA.</p

Validation of MLPA probes on 88 previously characterised mycobacterial strains.

<p>The MLPA was performed on 79 <i>M. tuberculosis</i> isolates (strains 1–79), nine non-tuberculosis mycobacteria (strains 80–88) and one species unrelated to mycobacteria (strain 89). Species identification was determined on the basis of the presence or absence of MLPA markers following calls mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043240#pone-0043240-g002" target="_blank">Figure 2</a>. Results obtained by MLPA were compared to results obtained from testing the same strain by other molecular methods. ^aStrain-specific drug resistance profiles and genotypic information obtained by other molecular methods is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043240#pone.0043240.s001" target="_blank">Table S1</a>. The presence or absence of an MLPA product is indicated with a black square or a white square, respectively. The confirmation of the MLPA result by other molecular methods is indicated with a green dot; conflicting results between MLPA and other molecular methods are indicated with a red cross. ND =  Analysis for this marker was not done. MTB4 is defined as <i>M. tuberculosis</i> group 4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043240#pone.0043240-Hershberg1" target="_blank">[26]</a> but not X family, LAM or Haarlem.</p

Dot plot of MLPA probe-specific MFI values of strains analysed.

<p>Median fluorescence intensity (MFI) values are indicated for each MLPA probe for every mycobacterial strain tested. The threshold used to call the presence or absence of a maker, MFI of 150, is indicated with a horizontal dashed line. Non-functional MLPA probes are indicated to the right side of the plot separated with a vertical dashed line. False positives or false negatives are highlighted in red. Brackets indicate whether a MLPA probe targets the wildtype sequence (wt), SNP (mut), the presence (P) or absence (A) of an RD, or a species-specific sequence (S).</p