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

Identification of Salmonella enterica serovar Typhi genotypes by use of rapid multiplex ligation-dependent probe amplification.

Salmonella enterica serovar Typhi, the causative agent of typhoid fever, is highly clonal and genetically conserved, making isolate subtyping difficult. We describe a standardized multiplex ligation-dependent probe amplification (MLPA) genotyping scheme targeting 11 key phylogenetic markers of the S. Typhi genome. The MLPA method demonstrated 90% concordance with single nucleotide polymorphism (SNP) typing, the gold standard for S. Typhi genotyping, and had the ability to identify isolates of the H58 haplotype, which is associated with resistance to multiple antimicrobials. Additionally, the assay permitted the detection of fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene gyrA and the topoisomerase gene parC with a sensitivity of 100%. The MLPA methodology is simple and reliable, providing phylogenetically and phenotypically relevant genotyping information. This MLPA scheme offers a more-sensitive and interpretable alternative to the nonphylogenetic subgrouping methodologies that are currently used in reference and research laboratories in areas where typhoid is endemic

Optimization of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins with V1/V2 Deleted, Using Virus Evolution▿

The human immunodeficiency virus type 1 envelope glycoprotein (Env) complex is the principal focus of neutralizing antibody-based vaccines. The functional Env complex is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The individual subunits have proven unsuccessful as vaccines presumably because they do not resemble the functional Env complex. Variable domains and carbohydrates shield vulnerable neutralization epitopes on the functional Env complex. The deletion of variable loops has been shown to improve gp120's immunogenicity; however, problems have been encountered when introducing such modifications in stabilized Env trimer constructs. To address these issues, we have created a set of V1/V2 and V3 loop deletion variants in the context of complete virus to allow optimization by forced virus evolution. Compensatory second-site substitutions included the addition and/or removal of specific carbohydrates, changes in the disulfide-bonded architecture of the V1/V2 stem, the replacement of hydrophobic residues by hydrophilic and charged residues, and changes in distal parts of gp120 and gp41. These viruses displayed increased sensitivity to neutralizing antibodies, demonstrating the improved exposure of conserved domains. The results show that we can select for functionally improved Env variants with loop deletions through forced virus evolution. Selected evolved Env variants were transferred to stabilized Env trimer constructs and were shown to improve trimer expression and secretion. Based on these findings, we can make recommendations on how to delete the V1/V2 domain from recombinant Env trimers for vaccine and X-ray crystallography studies. In general, virus evolution may provide a powerful tool to optimize Env vaccine antigens

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

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

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

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