Methods For Detecting Burkholderia pseudomallei and Burkholderia mallei RNA with RT-qPCR


Researchers at TGen and Northern Arizona University (NAU) have developed a method for detecting Burkholderia pseudomallei and Burkholderia mallei from the 16S ribosomal RNA in a sample using a Reverse Transcriptase quantitative PCR (RT-qPCR) assay.  This assay enables faster, more sensitive detection of B. pseudomallei and B. mallei in a variety of sample types for rapid diagnosis and treatment in a clinical setting.


Melioidosis is an infectious disease endemic to Southeast Asia and northern Australia caused by the Gram-negative bacterium B. pseudomallei, with estimated mortality rates in northeast Thailand of 50% and Australia of 19%.  Additionally, B. mallei is closely related to B. pseudomallei and causes additional serious infectious diseases.  These infectious diseases have long incubation periods, and have been poorly diagnosed in the past due to factors including the mimicking of other common bacteria infections and the low concentration of bacteria cells in common types of diagnostic samples.  With these challenges presented by pathogenic Burkholderia species,  the methods of microbiological culture, polysaccharide microarray, ELISA, and real time PCR assays targeting DNA from a Type III Secretion System gene (TTS1) have had difficulty achieving the requisite sensitivity and specificity for a rapid and accurate diagnosis, which delays treatment and increases the risk for patients.


The RT-qPCR assay developed by the researchers at TGen and NAU, based on a B. pseudomallei and B. mallei specific 16S hybridization real-time PCR assay, produced a 3-4 order of magnitude improvement in sensitivity for detecting B. pseudomallei  in a sample derived from laboratory cultures over a previously published real time assay that targets TTS1.  This RT-qPCR assay targeting 16S ribosomal RNA consistently amplified from about 10-13 Ct prior to the TTS1 assay for traditional and reverse transcriptase configurations respectively, indicating many more targets present for the TGen and NAU developed assay than for the TTS1 assay.  By increasing the number of available targets, this assay overcomes the previously known problem of detecting low levels of Burkholderia in blood samples.




Link to AU Issued patent AU 2013262783 A1


Link to US Published Patent Application US 2015/0167056 A1

Patent Information:
For Information, Contact:
Katie Bray
Intellectual Property Counsel
The Translational Genomics Research Institute
James Schupp
Rebecca Colman
Jordan Buchhagen
Paul Keim
David Engelthaler