Combined species identification, genotyping, and drug resistance detection of mycobacterium tuberculosis cultures by mlpa on a bead-based array

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management

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

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

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