A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Most Relevant Members of the <i>B</i>. <i>pseudomallei</i> Complex: <i>B</i>. <i>mallei</i>, <i>B</i>. <i>pseudomallei</i>, and <i>B</i>. <i>thailandensis</i>

<div><p>The <i>Burkholderia pseudomallei</i> complex classically consisted of <i>B</i>. <i>mallei</i>, <i>B</i>. <i>pseudomallei</i>, and <i>B</i>. <i>thailandensis</i>, but has now expanded to include <i>B</i>. <i>oklahomensis</i>, <i>B</i>. <i>humptydooensis</i>, and three unassigned <i>Burkholderia</i> clades. Methods for detecting and differentiating the <i>B</i>. <i>pseudomallei</i> complex has been the topic of recent research due to phenotypic and genotypic similarities of these species. <i>B</i>. <i>mallei</i> and <i>B</i>. <i>pseudomallei</i> are recognized as CDC Tier 1 select agents, and are the causative agents of glanders and melioidosis, respectively. Although <i>B</i>. <i>thailandensis</i> and <i>B</i>. <i>oklahomensis</i> are generally avirulent, both display similar phenotypic characteristics to that of <i>B</i>. <i>pseudomallei</i>. <i>B</i>. <i>humptydooensis</i> and the <i>Burkholderia</i> clades are genetically similar to the <i>B</i>. <i>pseudomallei</i> complex, and are not associated with disease. Optimal identification of these species remains problematic, and PCR-based methods can resolve issues with <i>B</i>. <i>pseudomallei</i> complex detection and differentiation. Currently, no PCR assay is available that detects the major species of the <i>B</i>. <i>pseudomallei</i> complex. A real-time PCR assay in a multiplex single-tube format was developed to simultaneously detect and differentiate <i>B</i>. <i>mallei</i>, <i>B</i>. <i>pseudomallei</i>, and <i>B</i>. <i>thailandensis</i>, and a common sequence found in <i>B</i>. <i>pseudomallei</i>, <i>B</i>. <i>mallei</i>, <i>B</i>. <i>thailandensis</i>, and <i>B</i>. <i>oklahomensis</i>. A total of 309 <i>Burkholderia</i> isolates and 5 other bacterial species were evaluated. The assay was 100% sensitive and specific, demonstrated sensitivity beyond culture and GC methods for the isolates tested, and is completed in about an hour with a detection limit between 2.6pg and 48.9pg of gDNA. Bioinformatic analyses also showed the assay is likely 100% specific and sensitive for all 84 fully sequenced <i>B</i>. <i>pseudomallei</i>, <i>B</i>. <i>mallei</i>, <i>B</i>. <i>thailandensis</i>, and <i>B</i>. <i>oklahomensis</i> strains currently available in GenBank. For these reasons, this assay could be a rapid and sensitive tool in the detection and differentiation for those species of the <i>B</i>. <i>pseudomallei</i> complex with recognized clinical and practical significance.</p></div

Sensitivity of the multiplex assay.

<p>Detection limits of the quadruplex assay and standard curves derived from serial dilutions of purified genomic DNAs for some species of the <i>B</i>. <i>pseudomallei</i> complex. (a) 16.5 kDa assay for <i>B</i>. <i>mallei</i>, (b) <i>orf11</i> assay for <i>B</i>. <i>pseudomallei</i>, (c) 70 kDa assay for <i>B</i>. <i>thailandensis</i>, (d) <i>fliC</i> assay for <i>B</i>. <i>mallei</i>, (e) <i>fliC</i> assay for <i>B</i>. <i>pseudomallei</i>, and (f) <i>fliC</i> assay for <i>B</i>. <i>thailandensis</i>.</p

Primer and probe sequences of the quadruplex assay.

<p>Primer and probe sequences of the quadruplex assay.</p

Sensitivity of the singleplex assays.

<p>Detection limits of the singleplex assays and standard curves derived from serial dilutions of purified genomic DNAs for some species of the <i>B</i>. <i>pseudomallei</i> complex. (a) 16.5 kDa assay for <i>B</i>. <i>mallei</i>, (b) <i>orf11</i> assay for <i>B</i>. <i>pseudomallei</i>, (c) 70 kDa for <i>B</i>. <i>thailandensis</i>, (d) <i>fliC</i> assay for <i>B</i>. <i>mallei</i>, (e) <i>fliC</i> assay for <i>B</i>. <i>pseudomallei</i>, and (f) <i>fliC</i> assay for <i>B</i>. <i>thailandensis</i>.</p