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

Erin P Price; J L Dale; J M Cook; Derek S Sarovich; M L Seymour; J L Ginther; E L Kaufman; S M Beckstrom-Sternberg; M Mayo; M Kaestli; M B Glass; J E Gee; V Wuthiekanun; J M Warner; A Baker; J T Foster; P Tan; A Tuanyok; D Limmathurotsakul; S J Peacock; B J Currie; D M Wagner; P Keim; T Pearson

doi:10.1371/journal.pone.0037723

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

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

Erin P Price, J L Dale, J M Cook, Derek S Sarovich, M L Seymour, J L Ginther, E L Kaufman, S M Beckstrom-Sternberg, M Mayo, M Kaestli, …

PLoS One, Vol.7(5), e37723

2012

DOI: https://doi.org/10.1371/journal.pone.0037723

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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. © 2012 Price et al.

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Title: Development and validation of burkholderia pseudomallei-specific real-time pcr assays for clinical, environmental or forensic detection applications
Authors: Erin P Price (Author) - Northern Arizona University, United States
J L Dale (Author) - Northern Arizona University, United States
J M Cook (Author) - Northern Arizona University, United States
Derek S Sarovich (Author) - Northern Arizona University, United States
M L Seymour (Author) - Northern Arizona University, United States
J L Ginther (Author) - Northern Arizona University, United States
E L Kaufman (Author) - Northern Arizona University, United States
S M Beckstrom-Sternberg (Author) - Northern Arizona University, United States
M Mayo (Author) - Menzies School of Health Research
M Kaestli (Author) - Menzies School of Health Research
M B Glass (Author) - Centres for Disease Control and Prevention, United States
J E Gee (Author) - Centres for Disease Control and Prevention, United States
V Wuthiekanun (Author) - Mahidol University, Thailand
J M Warner (Author) - James Cook University
A Baker (Author) - James Cook University
J T Foster (Author) - Northern Arizona University, United States
P Tan (Author) - Genome Institute of Singapore, Singapore
A Tuanyok (Author) - Northern Arizona University, United States
D Limmathurotsakul (Author) - Mahidol University, Thailand
S J Peacock (Author) - Mahidol University, Thailand
B J Currie (Author) - Menzies School of Health Research
D M Wagner (Author) - Northern Arizona University, United States
P Keim (Author) - Northern Arizona University, United States
T Pearson (Author) - Northern Arizona University, United States
Publication details: PLoS One, Vol.7(5), e37723
Publisher: Public Library of Science
Date published: 2012
DOI: 10.1371/journal.pone.0037723
ISSN: 1932-6203
Copyright note: Copyright © 2012 Price et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Organisation Unit: University of the Sunshine Coast, Queensland; Centre for Bioinnovation
Language: English
Record Identifier: 99451062402621
Output Type: Journal article

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