Comparison of seven DNA extraction kits based on the average crossing threshold (C<i>_T</i>) values, standard deviation and number of PCR positives using the TTS1 real-time PCR protocol.

<p>CFU/mL  =  Colony forming units per milliliter; SD  =  Standard deviation.</p

Average concentration and purity of DNA extraction performed by commercially available DNA extraction kits on <i>Burkholderia pseudomallei</i> spiked whole blood containing EDTA.

<p>Averages based on all blood specimens.</p

Anthrax-like cutaneous lesion on left check of 70-year-old male Florida resident.

<p>Anthrax-like cutaneous lesion on left check of 70-year-old male Florida resident.</p

<i>B</i>. <i>cereus</i> BcFL2013 capsule. India ink stain of <i>B</i>. <i>cereus</i> BcFL2013 after overnight growth in defibrinated horse blood.

<p><i>B</i>. <i>cereus</i> BcFL2013 capsule. India ink stain of <i>B</i>. <i>cereus</i> BcFL2013 after overnight growth in defibrinated horse blood.</p

Results of serological testing of specimens from 2013 Florida patient with anthrax-like cutaneous lesion.

<p>Results of serological testing of specimens from 2013 Florida patient with anthrax-like cutaneous lesion.</p

Summary of results for testing of <i>B</i>. <i>cereus</i> BcFL2013 compared to <i>B</i>. <i>cereus</i> G9241 and <i>B</i>. <i>anthracis</i> Ames.

<p>Summary of results for testing of <i>B</i>. <i>cereus</i> BcFL2013 compared to <i>B</i>. <i>cereus</i> G9241 and <i>B</i>. <i>anthracis</i> Ames.</p

<i>B. pseudomallei</i> 122018 and 266152 assays designed in this study.

a<p>Underlined nucleotides indicate the position of the SNP in the TaqMan probe; lowercase nucleotides indicate a deliberately incorporated 5′ flap to enhance amplification efficiency <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037723#pone.0037723-Afonina1" target="_blank">[43]</a>.</p>b<p>Based on <i>B. pseudomallei</i> K96243 genome (GenBank Accession numbers BX571965 and BX571966 for chromosomes 1 and 2, respectively) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037723#pone.0037723-Holden1" target="_blank">[14]</a>.</p>c<p>‘Other species’ refers specifically to <i>B. thailandensis</i>, <i>B. oklahomensis</i> and <i>B. thailandensis-</i>like spp. <i>B. mallei</i> and other <i>Burkholderia</i> spp. (e.g. <i>B. vietnamiensis</i>, <i>B. ubonensis</i> etc.) do not amplify according to <i>in silico</i> and wet-bench analyses.</p

<i>Burkholderia</i> spp. DNA specificity panel used in this study.

a<p>According to genotyping results generated in the current study, 16 S sequencing or MLST.</p>b<p>Previously identified as ‘<i>Burkholderia</i> spp.’ and renamed <i>Burkholderia thailandensis/B. thailandensis-</i>like<i>/B. oklahomensis</i> based on their genotyping outcomes in this study. However, we could not accurately differentiate these three species as they all amplify the non-<i>B. pseudomallei</i> probe in the 122018 and 266152 assays. Neither TTS1 nor BurkDiff assays amplify <i>B. thailandensis</i>, <i>B. thailandensis-</i>like or <i>B. oklahomensis</i> species, and thus could not be used for species assignment.</p>c<p>Ruled out as being <i>B. pseudomallei</i>, <i>B. thailandensis</i>, <i>B. thailandensis-</i>like, <i>B. oklahomensis</i>, <i>B. vietnamiensis</i> and <i>B. ubonensis</i> but strongly suspected to be <i>Burkholderia</i> spp. due to their amplification using <i>B. vietnamiensis</i> and <i>B. ubonensis</i>-specific assays (Price et al., unpublished data).</p

266152 TaqMan dual probe assay.

<p>Green, the <i>B. pseudomallei</i>-specific TaqMan probe preferentially amplifies <i>B. pseudomallei</i> template; the non-<i>B.pseudomallei</i> TaqMan probe (red) amplifies well in <i>B. thailandensis-</i>like species and weakly in <i>B. thailandensis</i> and <i>B. oklahomensis</i>. Other <i>Burkholderia</i> spp. do not amplify.</p

122018 TaqMan dual probe assay.

<p>Red, the <i>B. pseudomallei-</i>specific TaqMan probe amplifies only <i>B. pseudomallei</i> template; the non-<i>B.pseudomallei</i> TaqMan probe (green) amplifies well with <i>B. thailandensis</i> and <i>B. thailandensis-</i>like species and weakly with <i>B. oklahomensis</i> templates but not <i>B. pseudomallei</i>. Other <i>Burkholderia</i> spp. do not amplify with either probe.</p