A 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

• Published in: PloS one Vol. 11; no. 10; p. e0164006
• Main Authors: Lowe, Chinn-Woan, Satterfield, Benjamin A, Nelson, Daniel B, Thiriot, Joseph D, Heder, Michael J, March, Jordon K, Drake, David S, Lew, Cynthia S, Bunnell, Annette J, Moore, Emily S, O'Neill, Kim L, Robison, Richard A
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 13.10.2016; Public Library of Science (PLoS)
• Subjects: Assaying; Bacteria; Biology and Life Sciences; Burkholderia; Burkholderia - genetics; Burkholderia - isolation & purification; Burkholderia Infections - diagnosis; Burkholderia Infections - microbiology; Burkholderia mallei; Burkholderia mallei - genetics; Burkholderia mallei - isolation & purification; Burkholderia pseudomallei; Burkholderia pseudomallei - genetics; Burkholderia pseudomallei - isolation & purification; Differentiation; DNA, Bacterial - isolation & purification; Fatalities; Glanders; Glanders - microbiology; Humans; Identification methods; Infections; Infectious diseases; Laboratories; Medicine and Health Sciences; Melioidosis; Melioidosis - microbiology; Molecular biology; Oryza; Pathogens; Polymerase chain reaction; Real time; Real-Time Polymerase Chain Reaction - methods; Research and Analysis Methods; Sensitivity analysis; Sensitivity and Specificity; Sequence Analysis, DNA - methods; Species; Southeast Asia; Australia; Asia; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0164006

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.