Development and Validation of Burkholderia pseudomallei-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications

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

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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. 
    Original languageEnglish
    Article numbere37723
    Pages (from-to)1-9
    Number of pages9
    JournalPLoS One
    Volume7
    Issue number5
    DOIs
    Publication statusPublished - 2012

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    Burkholderia pseudomallei
    Real-Time Polymerase Chain Reaction
    Assays
    quantitative polymerase chain reaction
    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

    Cite this

    Price, Erin ; Dale, Julie ; Cook, James ; Sarovich, Derek ; Seymour, Meagan ; Ginther, Jennifer L ; Kaufman, L ; Beckstrom-Sternberg, Stephen ; Mayo, Mark ; Kaestli, Mirjam ; Glass, Mindy ; Gee, J ; Wuthiekanun, Vanaporn ; Warner, Jeffrey ; Baker, Anthony ; Foster, Jeffrey ; TAN, P ; Tuanyok, Apichai ; Limmathurotsakul, Direk ; Peacock, Sharon J ; Currie, Bart ; Wagner, David M ; Keim, Paul S ; Pearson, Talima. / 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. pp. 1-9.
    @article{d280625ed31f4b12a04f4a3277835fdb,
    title = "Development and Validation of Burkholderia pseudomallei-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications",
    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. ",
    keywords = "bacterial DNA, article, BukDiff assay, Burkholderia, Burkholderia oklahomensis, Burkholderia pseudomallei, Burkholderia thailandensis, clinical practice, DNA determination, environment, forensic identification, fungal strain, intermethod comparison, molecular probe, nonhuman, quantitative study, reverse transcription polymerase chain reaction, sensitivity analysis, TTS1 assay, validation study, comparative study, DNA sequence, genetics, melioidosis, methodology, microbiology, real time polymerase chain reaction, single nucleotide polymorphism, species difference, Bacteria (microorganisms), Burkholderiaceae, Melioidosis, Polymorphism, Single Nucleotide, Real-Time Polymerase Chain Reaction, Sequence Analysis, DNA, Species Specificity",
    author = "Erin Price and Julie Dale and James Cook and Derek Sarovich and Meagan Seymour and Ginther, {Jennifer L} and L Kaufman and Stephen Beckstrom-Sternberg and Mark Mayo and Mirjam Kaestli and Mindy Glass and J Gee and Vanaporn Wuthiekanun and Jeffrey Warner and Anthony Baker and Jeffrey Foster and P TAN and Apichai Tuanyok and Direk Limmathurotsakul and Peacock, {Sharon J} and Bart Currie and Wagner, {David M} and Keim, {Paul S} and Talima Pearson",
    year = "2012",
    doi = "10.1371/journal.pone.0037723",
    language = "English",
    volume = "7",
    pages = "1--9",
    journal = "PLoS One",
    issn = "1932-6203",
    publisher = "Public Library of Science (PLoS)",
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    Price, E, Dale, J, Cook, J, Sarovich, D, Seymour, M, Ginther, JL, Kaufman, L, Beckstrom-Sternberg, S, Mayo, M, Kaestli, M, Glass, M, Gee, J, Wuthiekanun, V, Warner, J, Baker, A, Foster, J, TAN, P, Tuanyok, A, Limmathurotsakul, D, Peacock, SJ, Currie, B, Wagner, DM, Keim, PS & Pearson, T 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, pp. 1-9. https://doi.org/10.1371/journal.pone.0037723

    Development and Validation of Burkholderia pseudomallei-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications. / Price, Erin; Dale, Julie; Cook, James; Sarovich, Derek; Seymour, Meagan; Ginther, Jennifer L; Kaufman, L; Beckstrom-Sternberg, Stephen; Mayo, Mark; Kaestli, Mirjam; Glass, Mindy; Gee, J; Wuthiekanun, Vanaporn; Warner, Jeffrey; Baker, Anthony; Foster, Jeffrey; TAN, P; Tuanyok, Apichai; Limmathurotsakul, Direk; Peacock, Sharon J; Currie, Bart; Wagner, David M; Keim, Paul S; Pearson, Talima.

    In: PLoS One, Vol. 7, No. 5, e37723, 2012, p. 1-9.

    Research output: Contribution to journalArticleResearchpeer-review

    TY - JOUR

    T1 - Development and Validation of Burkholderia pseudomallei-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications

    AU - Price, Erin

    AU - Dale, Julie

    AU - Cook, James

    AU - Sarovich, Derek

    AU - Seymour, Meagan

    AU - Ginther, Jennifer L

    AU - Kaufman, L

    AU - Beckstrom-Sternberg, Stephen

    AU - Mayo, Mark

    AU - Kaestli, Mirjam

    AU - Glass, Mindy

    AU - Gee, J

    AU - Wuthiekanun, Vanaporn

    AU - Warner, Jeffrey

    AU - Baker, Anthony

    AU - Foster, Jeffrey

    AU - TAN, P

    AU - Tuanyok, Apichai

    AU - Limmathurotsakul, Direk

    AU - Peacock, Sharon J

    AU - Currie, Bart

    AU - Wagner, David M

    AU - Keim, Paul S

    AU - Pearson, Talima

    PY - 2012

    Y1 - 2012

    N2 - 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. 

    AB - 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. 

    KW - bacterial DNA

    KW - article

    KW - BukDiff assay

    KW - Burkholderia

    KW - Burkholderia oklahomensis

    KW - Burkholderia pseudomallei

    KW - Burkholderia thailandensis

    KW - clinical practice

    KW - DNA determination

    KW - environment

    KW - forensic identification

    KW - fungal strain

    KW - intermethod comparison

    KW - molecular probe

    KW - nonhuman

    KW - quantitative study

    KW - reverse transcription polymerase chain reaction

    KW - sensitivity analysis

    KW - TTS1 assay

    KW - validation study

    KW - comparative study

    KW - DNA sequence

    KW - genetics

    KW - melioidosis

    KW - methodology

    KW - microbiology

    KW - real time polymerase chain reaction

    KW - single nucleotide polymorphism

    KW - species difference

    KW - Bacteria (microorganisms)

    KW - Burkholderiaceae

    KW - Melioidosis

    KW - Polymorphism, Single Nucleotide

    KW - Real-Time Polymerase Chain Reaction

    KW - Sequence Analysis, DNA

    KW - Species Specificity

    U2 - 10.1371/journal.pone.0037723

    DO - 10.1371/journal.pone.0037723

    M3 - Article

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    SP - 1

    EP - 9

    JO - PLoS One

    JF - PLoS One

    SN - 1932-6203

    IS - 5

    M1 - e37723

    ER -