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.

Autor:	Lowe CW; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., Satterfield BA; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., Nelson DB; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., Thiriot JD; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., Heder MJ; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., March JK; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., Drake DS; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., Lew CS; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., Bunnell AJ; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., Moore ES; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., O'Neill KL; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America., Robison RA; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.
Jazyk:	angličtina
Zdroj:	PloS one [PLoS One] 2016 Oct 13; Vol. 11 (10), pp. e0164006. Date of Electronic Publication: 2016 Oct 13 (Print Publication: 2016).
DOI:	10.1371/journal.pone.0164006
Abstrakt:	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. Competing Interests: The authors have declared that no competing interests exist.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu Plný text ve formátu PDF