Phenotypic effects of the <i>oacA</i> mutation in <i>B. pseudomallei</i> 112 and <i>B. thailandensis</i> TXDOH revealed by immunoblot analysis.

<p>LPS samples from <i>B. pseudomallei</i> K96243 and 112, <i>B. mallei</i> ATCC23344, and <i>B. thailandensis</i> E264 and TXDOH, Lanes 1–5, respectively, hybridized against serotype A patient's serum (panel A), and <i>B. mallei</i> LPS-specific mAb 3D11 (panel B). As predicted, LPS samples from <i>B. pseudomallei</i> 112 (lane 2) and <i>B. thailandensis</i> TXDOH (lane 5) were strongly positive to the mAb 3D11 due to the mutation of their <i>oacA</i> genes. Lane L is a pre-stained protein standard ladder.</p

Diversity of <i>B. pseudomallei</i> LPS banding patterns and their serological specificity.

<p>Panel A is silver strained SDS-PAGE of four different LPS phenotypes; panels B and C are immunoblotting analysis of the same LPS samples using sera from melioidosis patients with known infection by LPS genotype A, or B strains, respectively. Lanes 1–4 are typical (type A), atypical (type B), a novel atypical (type B variant or type B2), and rough LPS types, respectively; lane L is a pre-stained protein standard ladder. We note that the typical LPS was specifically seroreactive to the antibody from patient who was infected by LPS genotype A strain, whereas, the atypical LPS types (lanes 2 and 3) were seroreactive with the antibody from the LPS genotype B infected patient only. Rough LPS or no-banding LPS appearance (lane 4) was seronegative to both sera.</p

Genotyping scheme and frequencies of three different LPS genotypes identified in <i>B. pseudomallei</i> populations.

<p>Multiplex SYBR-Green PCR assays were developed to target the presence of genes: <i>wbiE</i>, BUC_3396, and BURP840_LPSb16, which were the representatives of LPS genotypes A, B, and B2, respectively. PCR amplicons from these 3 gene targets were differentiated by melting dissociation (A); or sizing (B); lanes 1, 2, 3, and 4 are PCR products from strains K96243, 576, MSHR840, and non-DNA template control (NTC), respectively; and L, 1 kb-plus DNA ladder. We note that LPS genotype A was the most common LPS genotype, whereas a majority of the LPS genotype B was found in strains from Australia (approx.13.8%). Genotype B2 was found in strains from Australia and Papua New Guinea (PNG) only (C).</p

Differential LPS phenotypes and serum susceptibility of the chronic lung strains.

<p>Panel A demonstrates LPS phenotypes based upon SDS-PAGE analysis of select chronic lung strains; lanes 1–9, LPS samples from the chronic lung strains MSHR1043, MSHR1048, MSHR1218, MSHR1288, MSHR1290, MSHR1418, MSHR1459, MSHR1655, and MSHR3042, respectively; L, protein standard ladder. Panel B shows differential serum susceptibility in four select chronic lung <i>B. pseudomallei</i> strains grown in 30% of normal human serum (NHS); a well-known serum resistant <i>B. pseudomallei</i> strain 1026b, and a laboratory <i>E. coli</i> strain HB101 were used as the positive and negative controls in this study, respectively. We note that strains MSHR1655 and MSHR3042, the rough LPS strains that had mutation in their <i>wbiI</i> genes were unable to multiply in the presence of 30% NHS, whereas, the typical LPS strains MSHR1043 and MSHR1048 from the same patient were able to utilize the NHS as nutrients.</p

Point mutations found in <i>wbiI</i> and <i>oacA</i> genes in clonal <i>B. pseudomallei</i> strains.

<p>These strains were collected chronologically from a single chronic lung patient who had severe bronchiectasis associated with melioidosis over almost 8 years. Panel A is the chronological order of these <i>B. pseudomallei</i> strains. Panel B demonstrates an extra base (“G”) that was found to cause frame-shift mutation in <i>wbiI</i> gene of all <i>B. pseudomallei</i> strains collected from day 550 onward. Panel C demonstrates the insertion of two extra bases “TC” in BPSL1936, the <i>oacA</i> homolog, in the same strains that had the <i>wbiI</i> mutation. Note: the <i>wbiI</i> gene of <i>B. pseudomallei</i> K96243 and <i>oacA</i> gene of <i>B. thailandensis</i> E264 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001453#pntd.0001453-Brett1" target="_blank">[14]</a> were used as comparisons.</p

Melt-MAMAs targeting specific groups within eight pathogen species.

a<p>First design attempt and altered primer ratio optimization.</p>b<p>Success after combining first or second design attempts and altered primer ratio optimization.</p>c<p>Failed after first design attempt.</p>d<p>Assays that required altered primer concentration ratios.</p

Real-time PCR amplification and dissociation (melt) curve plots.

<p><i>B. anthracis</i> Melt-MAMA SYBR® Green assay targeting the A.Br.004 genetic clade. (A & C) The amplification of two alleles are illustrated for haploid template (<i>Bacillus anthracis</i>) possessing an ‘A’ polymorphic SNP-state or ‘G’ state. Each amplification plot represents a single PCR reaction containing a reverse “common” primer and two allele-specific MAMA primers. The AS-MAMA primers anneal to the same template target and then compete for extension across the SNP position. The polymerase-mediated extension rate of the 3′match AS-MAMA primer (perfect primer-template complex) exceeds that of the 3′mismatched MAMA primer (mismatched primer-template complex), thus the perfect match primer-template complex outcompetes the mismatched primer-template complex and dominates the PCR amplification. (B & D) Plots of the temperature-dissociation (melt) curve of the final PCR products for the two allele templates are shown next to their respective amplification plots (green arrows). Allele-specific PCR products are easily differentiated through temperature-dissociation (melt) curve analysis, which is conferred by the GC-clamp engineered on one of the AS-MAMA primer.</p

Genotyping over a broad range of DNA amounts.

<p>Melt-MAMA genotyping accuracy is not diminished at lower amounts of DNA, even at near-single copy for some assays. The sensitivity of individual melt-MAMAs varies greatly. This <i>B. anthracis</i> melt-MAMA (A.Br.003 clade) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a> accurately genotyped DNA regardless of starting amounts as long as it was sufficient to support amplification. (A & B) The respective amplification plots of genomic DNA of ‘G’ allele and ‘A’ SNP allele templates show the amplification curves of templates titrated in ten-fold serial dilutions and in replicates of eight. The number assigned to each amplification curve (1–8) denotes the DNA amount for the starting template. (C) The temperature-dissociation (melt) curve derivatives for all initial template amounts are shown (numbers denote DNA amount shown). This panel illustrates that genotyping accuracy was not affected by DNA amounts, even at near-single copy levels. Similar to TaqMan assays, the detection of low levels of DNA template by Melt-MAMA is also subject to stochastic sampling effects (<i>B. anthracis</i> single copy ∼6 fg), which is predictable using a Poisson distribution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a>.</p

Melt-MAMA validation work flow.

<p>This figure shows the sequential steps involved in validation of Melt-MAMA assays. After SNP selection (step I), Melt-MAMA are designed so that the amplicon is <100 bp in length (step II). Assays are screened across ancestral and derived DNA templates under 3 primer ratio conditions where 1∶1 represents equal primer ratio, 4∶1 represents ancestral primer 4x and derived primer 1x, and 1∶4 represents ancestral primer 1x and derived primer 4x (step III). Five outcomes are indicated (step III a–e). Based on the performance of <i>B. anthracis</i>, <i>F. tularensis</i>, and <i>Y. pestis</i> assays, 70–80% Melt-MAMAs accurately genotyped at one of the tested primer ratio condition (step IIIa). These successful assays were immediately screened on a diversity panel of DNA samples (step IV). The remaining assays (20–30%) resulted in one of the other four outcomes (step III b–e). Each outcome required additional specific validation steps to determine the optimal PCR conditions or the need to abandon the SNP altogether. Our overall design success rate increased from 46% to 87%.</p

TaqMan assay performance at a broad range of DNA amounts.

<p>A <i>B. anthracis</i> TaqMan assay was used to screen the polymorphic ‘G’ or ‘A’ DNA templates (ancestral and derived, respectively) used in the <i>B. anthracis</i> Melt-MAMAs. (A & B) The respective amplification plots of genomic DNA of ‘G’ allele and ‘A’ SNP allele templates show the amplification curves of templates titrated in ten-fold serial dilutions and in replicates of eight. The number assigned to each amplification curve (1–9) denotes the DNA amount for the starting template. (C) Both genomic template types were of equal amounts. The consistency of amplification dropped with lower amounts of initial template, but the dilution levels containing less than a single copy (<i>B. anthracis</i> single copy ∼6 fg) was still detectable in some reactions. Detection of low-level DNA template by TaqMan assays is subject to stochastic sampling effects, which is predictable using a Poisson distribution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a>.</p