Development and validation of burkholderia pseudomallei-specific real-time pcr assays for clinical, environmental or forensic detection applications

Erin P. Price, Julia L. Dale, James M. Cook, Derek S. Sarovich, Meagan L. Seymour, Jennifer L. Ginther, Emily L. Kaufman, Stephen M Beckstrom-Sternberg, Mark Mayo, Mirjam Kaestli, Mindy B. Glass, Jay E. Gee, Vanaporn Wuthiekanun, Jeffrey M. Warner, Anthony Baker, Jeffrey T Foster, Patrick Tan, Apichai Tuanyok, Direk Limmathurotsakul, Sharon J. Peacock & 4 others Bart J. Currie, David M Wagner, Paul S Keim, Talima R Pearson

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (&2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.

Original languageEnglish (US)
Article numbere37723
JournalPLoS One
Volume7
Issue number5
DOIs
StatePublished - May 18 2012

Fingerprint

Burkholderia pseudomallei
Assays
assays
Burkholderiaceae
Quality Control
quality control
Quality control
Melioidosis
Burkholderia
forensic sciences
Species Specificity
Diagnostic Errors
Computer Simulation
Genetic Recombination
Limit of Detection
Bacteria
detection limit

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Development and validation of burkholderia pseudomallei-specific real-time pcr assays for clinical, environmental or forensic detection applications. / Price, Erin P.; Dale, Julia L.; Cook, James M.; Sarovich, Derek S.; Seymour, Meagan L.; Ginther, Jennifer L.; Kaufman, Emily L.; Beckstrom-Sternberg, Stephen M; Mayo, Mark; Kaestli, Mirjam; Glass, Mindy B.; Gee, Jay E.; Wuthiekanun, Vanaporn; Warner, Jeffrey M.; Baker, Anthony; Foster, Jeffrey T; Tan, Patrick; Tuanyok, Apichai; Limmathurotsakul, Direk; Peacock, Sharon J.; Currie, Bart J.; Wagner, David M; Keim, Paul S; Pearson, Talima R.

In: PLoS One, Vol. 7, No. 5, e37723, 18.05.2012.

Research output: Contribution to journalArticle

Price, EP, Dale, JL, Cook, JM, Sarovich, DS, Seymour, ML, Ginther, JL, Kaufman, EL, Beckstrom-Sternberg, SM, Mayo, M, Kaestli, M, Glass, MB, Gee, JE, Wuthiekanun, V, Warner, JM, Baker, A, Foster, JT, Tan, P, Tuanyok, A, Limmathurotsakul, D, Peacock, SJ, Currie, BJ, Wagner, DM, Keim, PS & Pearson, TR 2012, 'Development and validation of burkholderia pseudomallei-specific real-time pcr assays for clinical, environmental or forensic detection applications', PLoS One, vol. 7, no. 5, e37723. https://doi.org/10.1371/journal.pone.0037723
Price, Erin P. ; Dale, Julia L. ; Cook, James M. ; Sarovich, Derek S. ; Seymour, Meagan L. ; Ginther, Jennifer L. ; Kaufman, Emily L. ; Beckstrom-Sternberg, Stephen M ; Mayo, Mark ; Kaestli, Mirjam ; Glass, Mindy B. ; Gee, Jay E. ; Wuthiekanun, Vanaporn ; Warner, Jeffrey M. ; Baker, Anthony ; Foster, Jeffrey T ; Tan, Patrick ; Tuanyok, Apichai ; Limmathurotsakul, Direk ; Peacock, Sharon J. ; Currie, Bart J. ; Wagner, David M ; Keim, Paul S ; Pearson, Talima R. / Development and validation of burkholderia pseudomallei-specific real-time pcr assays for clinical, environmental or forensic detection applications. In: PLoS One. 2012 ; Vol. 7, No. 5.
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abstract = "The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (&2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100{\%} specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.",
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AU - Sarovich, Derek S.

AU - Seymour, Meagan L.

AU - Ginther, Jennifer L.

AU - Kaufman, Emily L.

AU - Beckstrom-Sternberg, Stephen M

AU - Mayo, Mark

AU - Kaestli, Mirjam

AU - Glass, Mindy B.

AU - Gee, Jay E.

AU - Wuthiekanun, Vanaporn

AU - Warner, Jeffrey M.

AU - Baker, Anthony

AU - Foster, Jeffrey T

AU - Tan, Patrick

AU - Tuanyok, Apichai

AU - Limmathurotsakul, Direk

AU - Peacock, Sharon J.

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AU - Wagner, David M

AU - Keim, Paul S

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