Open AccessA Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Most Relevant Members of the B. pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis
Author(s) -
Lowe Chinn-Woan,
Benjamin A. Satterfield,
Daniel B. Nelson,
Joseph Thiriot,
Michael J. Heder,
Jordon K. March,
David S. Drake,
Cynthia S. Lew,
Annette J Bunnell,
Emily S. Moore,
Kim L. O’Neill,
Richard A. Robison
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0164006
Subject(s) - burkholderia pseudomallei , melioidosis , biology , microbiology and biotechnology , burkholderia , polymerase chain reaction , virology , bacteria , genetics , gene
The Burkholderia pseudomallei complex classically consisted of B . mallei , B . pseudomallei , and B . thailandensis , but has now expanded to include B . oklahomensis , B . humptydooensis , and three unassigned Burkholderia clades. Methods for detecting and differentiating the B . pseudomallei complex has been the topic of recent research due to phenotypic and genotypic similarities of these species. B . mallei and B . pseudomallei are recognized as CDC Tier 1 select agents, and are the causative agents of glanders and melioidosis, respectively. Although B . thailandensis and B . oklahomensis are generally avirulent, both display similar phenotypic characteristics to that of B . pseudomallei . B . humptydooensis and the Burkholderia clades are genetically similar to the B . pseudomallei complex, and are not associated with disease. Optimal identification of these species remains problematic, and PCR-based methods can resolve issues with B . pseudomallei complex detection and differentiation. Currently, no PCR assay is available that detects the major species of the B . pseudomallei complex. A real-time PCR assay in a multiplex single-tube format was developed to simultaneously detect and differentiate B . mallei , B . pseudomallei , and B . thailandensis , and a common sequence found in B . pseudomallei , B . mallei , B . thailandensis , and B . oklahomensis . A total of 309 Burkholderia 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 B . pseudomallei , B . mallei , B . thailandensis , and B . oklahomensis 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 B . pseudomallei complex with recognized clinical and practical significance.