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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.
PLoS One. 2016; 11(10):e0164006.Plos

Abstract

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.

Authors+Show Affiliations

Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, 84602, United States of America.

Pub Type(s)

Evaluation Study
Journal Article

Language

eng

PubMed ID

27736903

Citation

Lowe, Chinn-Woan, et al. "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." PloS One, vol. 11, no. 10, 2016, pp. e0164006.
Lowe CW, Satterfield BA, Nelson DB, et al. 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. PLoS One. 2016;11(10):e0164006.
Lowe, C. W., Satterfield, B. A., Nelson, D. B., Thiriot, J. D., Heder, M. J., March, J. K., Drake, D. S., Lew, C. S., Bunnell, A. J., Moore, E. S., O'Neill, K. L., & Robison, R. A. (2016). 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. PloS One, 11(10), e0164006. https://doi.org/10.1371/journal.pone.0164006
Lowe CW, et al. 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. PLoS One. 2016;11(10):e0164006. PubMed PMID: 27736903.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Lowe,Chinn-Woan, AU - Satterfield,Benjamin A, AU - Nelson,Daniel B, AU - Thiriot,Joseph D, AU - Heder,Michael J, AU - March,Jordon K, AU - Drake,David S, AU - Lew,Cynthia S, AU - Bunnell,Annette J, AU - Moore,Emily S, AU - O'Neill,Kim L, AU - Robison,Richard A, Y1 - 2016/10/13/ PY - 2015/10/11/received PY - 2016/09/19/accepted PY - 2016/10/14/entrez PY - 2016/10/14/pubmed PY - 2017/5/23/medline SP - e0164006 EP - e0164006 JF - PloS one JO - PLoS One VL - 11 IS - 10 N2 - 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/27736903/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_ L2 - https://dx.plos.org/10.1371/journal.pone.0164006 DB - PRIME DP - Unbound Medicine ER -