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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
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 bee...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063335/ https://www.ncbi.nlm.nih.gov/pubmed/27736903 http://dx.doi.org/10.1371/journal.pone.0164006 |
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author | 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. |
author_facet | 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. |
author_sort | Lowe, Chinn-Woan |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5063335 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-50633352016-11-04 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 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. PLoS One Research Article 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. Public Library of Science 2016-10-13 /pmc/articles/PMC5063335/ /pubmed/27736903 http://dx.doi.org/10.1371/journal.pone.0164006 Text en © 2016 Lowe et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article 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. 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 |
title | 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 |
title_full | 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 |
title_fullStr | 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 |
title_full_unstemmed | 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 |
title_short | 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 |
title_sort | 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 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063335/ https://www.ncbi.nlm.nih.gov/pubmed/27736903 http://dx.doi.org/10.1371/journal.pone.0164006 |
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