Development and Validation of Burkholderia pseudomallei-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications
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{"title"=>"Development and validation of burkholderia pseudomallei-specific real-time pcr assays for clinical, environmental or forensic detection applications", "type"=>"journal", "authors"=>[{"first_name"=>"Erin P.", "last_name"=>"Price", "scopus_author_id"=>"10639892400"}, {"first_name"=>"Julia L.", "last_name"=>"Dale", "scopus_author_id"=>"35409050600"}, {"first_name"=>"James M.", "last_name"=>"Cook", "scopus_author_id"=>"55023939800"}, {"first_name"=>"Derek S.", "last_name"=>"Sarovich", "scopus_author_id"=>"16231349000"}, {"first_name"=>"Meagan L.", "last_name"=>"Seymour", "scopus_author_id"=>"55018056900"}, {"first_name"=>"Jennifer L.", "last_name"=>"Ginther", "scopus_author_id"=>"35723582600"}, {"first_name"=>"Emily L.", "last_name"=>"Kaufman", "scopus_author_id"=>"55102639800"}, {"first_name"=>"Stephen M.", "last_name"=>"Beckstrom-Sternberg", "scopus_author_id"=>"6602727821"}, {"first_name"=>"Mark", "last_name"=>"Mayo", "scopus_author_id"=>"7202350361"}, {"first_name"=>"Mirjam", "last_name"=>"Kaestli", "scopus_author_id"=>"14066126400"}, {"first_name"=>"Mindy B.", "last_name"=>"Glass", "scopus_author_id"=>"7103225281"}, {"first_name"=>"Jay E.", "last_name"=>"Gee", "scopus_author_id"=>"35302513800"}, {"first_name"=>"Vanaporn", "last_name"=>"Wuthiekanun", "scopus_author_id"=>"7003699771"}, {"first_name"=>"Jeffrey M.", "last_name"=>"Warner", "scopus_author_id"=>"16551205300"}, {"first_name"=>"Anthony", "last_name"=>"Baker", "scopus_author_id"=>"37070199900"}, {"first_name"=>"Jeffrey T.", "last_name"=>"Foster", "scopus_author_id"=>"7403380491"}, {"first_name"=>"Patrick", "last_name"=>"Tan", "scopus_author_id"=>"34769387900"}, {"first_name"=>"Apichai", "last_name"=>"Tuanyok", "scopus_author_id"=>"6506832663"}, {"first_name"=>"Direk", "last_name"=>"Limmathurotsakul", "scopus_author_id"=>"9637805700"}, {"first_name"=>"Sharon J.", "last_name"=>"Peacock", "scopus_author_id"=>"7006230636"}, {"first_name"=>"Bart J.", "last_name"=>"Currie", "scopus_author_id"=>"14520829200"}, {"first_name"=>"David M.", "last_name"=>"Wagner", "scopus_author_id"=>"7401982601"}, {"first_name"=>"Paul", "last_name"=>"Keim", "scopus_author_id"=>"7007034048"}, {"first_name"=>"Talima", "last_name"=>"Pearson", "scopus_author_id"=>"7202069259"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84862095063", "doi"=>"10.1371/journal.pone.0037723", "issn"=>"19326203", "pui"=>"364850196", "isbn"=>"1932-6203 (Electronic)\\n1932-6203 (Linking)", "pmid"=>"22624061", "scopus"=>"2-s2.0-84862095063"}, "id"=>"ed01292c-e8f7-390a-9128-73dc794f452e", "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.", "link"=>"http://www.mendeley.com/research/development-validation-burkholderia-pseudomalleispecific-realtime-pcr-assays-clinical-environmental", "reader_count"=>34, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Researcher"=>8, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>11, "Student > Postgraduate"=>1, "Student > Master"=>4, "Student > Bachelor"=>2, "Lecturer > Senior Lecturer"=>3, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Researcher"=>8, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>11, "Student > Postgraduate"=>1, "Student > Master"=>4, "Student > Bachelor"=>2, "Lecturer > Senior Lecturer"=>3, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>5, "Agricultural and Biological Sciences"=>13, "Medicine and Dentistry"=>7, "Veterinary Science and Veterinary Medicine"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>2, "Psychology"=>1, "Social Sciences"=>1, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>7}, "Social Sciences"=>{"Social Sciences"=>1}, "Psychology"=>{"Psychology"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>13}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}, "Unspecified"=>{"Unspecified"=>2}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>2}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>1}}, "reader_count_by_country"=>{"United States"=>1, "Malaysia"=>1, "Thailand"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/637113"], "description"=>"a<p>According to genotyping results generated in the current study, 16 S sequencing or MLST.</p>b<p>Previously identified as ‘<i>Burkholderia</i> spp.’ and renamed <i>Burkholderia thailandensis/B. thailandensis-</i>like<i>/B. oklahomensis</i> based on their genotyping outcomes in this study. However, we could not accurately differentiate these three species as they all amplify the non-<i>B. pseudomallei</i> probe in the 122018 and 266152 assays. Neither TTS1 nor BurkDiff assays amplify <i>B. thailandensis</i>, <i>B. thailandensis-</i>like or <i>B. oklahomensis</i> species, and thus could not be used for species assignment.</p>c<p>Ruled out as being <i>B. pseudomallei</i>, <i>B. thailandensis</i>, <i>B. thailandensis-</i>like, <i>B. oklahomensis</i>, <i>B. vietnamiensis</i> and <i>B. ubonensis</i> but strongly suspected to be <i>Burkholderia</i> spp. due to their amplification using <i>B. vietnamiensis</i> and <i>B. ubonensis</i>-specific assays (Price et al., unpublished data).</p>", "links"=>[], "tags"=>["dna", "specificity"], "article_id"=>307580, "categories"=>["Cell Biology", "Microbiology", "Infectious Diseases", "Biotechnology", "Biological Sciences", "Genetics", "Evolutionary Biology"], "users"=>["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", "Bart J. Currie", "David M. Wagner", "Paul Keim", "Talima Pearson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0037723.t002", "stats"=>{"downloads"=>3, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Burkholderia_spp_DNA_specificity_panel_used_in_this_study_/307580", "title"=>"<i>Burkholderia</i> spp. DNA specificity panel used in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-05-18 02:06:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/637047"], "description"=>"a<p>Underlined nucleotides indicate the position of the SNP in the TaqMan probe; lowercase nucleotides indicate a deliberately incorporated 5′ flap to enhance amplification efficiency <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037723#pone.0037723-Afonina1\" target=\"_blank\">[43]</a>.</p>b<p>Based on <i>B. pseudomallei</i> K96243 genome (GenBank Accession numbers BX571965 and BX571966 for chromosomes 1 and 2, respectively) <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037723#pone.0037723-Holden1\" target=\"_blank\">[14]</a>.</p>c<p>‘Other species’ refers specifically to <i>B. thailandensis</i>, <i>B. oklahomensis</i> and <i>B. thailandensis-</i>like spp. <i>B. mallei</i> and other <i>Burkholderia</i> spp. (e.g. <i>B. vietnamiensis</i>, <i>B. ubonensis</i> etc.) do not amplify according to <i>in silico</i> and wet-bench analyses.</p>", "links"=>[], "tags"=>["122018", "266152", "assays", "designed"], "article_id"=>307507, "categories"=>["Cell Biology", "Microbiology", "Infectious Diseases", "Biotechnology", "Biological Sciences", "Genetics", "Evolutionary Biology"], "users"=>["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", "Bart J. Currie", "David M. Wagner", "Paul Keim", "Talima Pearson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0037723.t001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_B_pseudomallei_122018_and_266152_assays_designed_in_this_study_/307507", "title"=>"<i>B. pseudomallei</i> 122018 and 266152 assays designed in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-05-18 02:05:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/329226", "https://ndownloader.figshare.com/files/329384", "https://ndownloader.figshare.com/files/329478", "https://ndownloader.figshare.com/files/329569", "https://ndownloader.figshare.com/files/329639", "https://ndownloader.figshare.com/files/329712", "https://ndownloader.figshare.com/files/329775", "https://ndownloader.figshare.com/files/329830", "https://ndownloader.figshare.com/files/329889", "https://ndownloader.figshare.com/files/329932", "https://ndownloader.figshare.com/files/329977", "https://ndownloader.figshare.com/files/330020"], "description"=>"<div><p>The bacterium <em>Burkholderia pseudomallei</em> 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 <em>B. pseudomallei</em> 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 <em>B. pseudomallei</em> from other Burkholderiaceae. Currently available molecular assays for <em>B. pseudomallei</em> detection lack rigorous validation across large <em>in silico</em> 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 <em>B. pseudomallei</em> specific assays, 122018 and 266152, using a dual-probe approach to differentiate <em>B. pseudomallei</em> from <em>B. thailandensis</em>, <em>B. oklahomensis</em> and <em>B. thailandensis</em>-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples) comprising <em>Burkholderia</em> spp. and non<em>-Burkholderia</em> bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published <em>B. pseudomallei</em>-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 <em>B. pseudomallei</em> as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated <em>B. pseudomallei</em> assays for the scientific research community.</p> </div>", "links"=>[], "tags"=>["validation", "Real-time", "pcr", "assays", "forensic", "detection", "applications"], "article_id"=>124996, "categories"=>["Cancer", "Cell Biology", "Microbiology", "Biotechnology", "Biological Sciences", "Genetics", "Evolutionary Biology"], "users"=>["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", "Bart J. Currie", "David M. Wagner", "Paul Keim", "Talima Pearson"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0037723.s001", "https://dx.doi.org/10.1371/journal.pone.0037723.s002", "https://dx.doi.org/10.1371/journal.pone.0037723.s003", "https://dx.doi.org/10.1371/journal.pone.0037723.s004", "https://dx.doi.org/10.1371/journal.pone.0037723.s005", "https://dx.doi.org/10.1371/journal.pone.0037723.s006", "https://dx.doi.org/10.1371/journal.pone.0037723.s007", "https://dx.doi.org/10.1371/journal.pone.0037723.s008", "https://dx.doi.org/10.1371/journal.pone.0037723.s009", "https://dx.doi.org/10.1371/journal.pone.0037723.s010", "https://dx.doi.org/10.1371/journal.pone.0037723.s011", "https://dx.doi.org/10.1371/journal.pone.0037723.s012"], "stats"=>{"downloads"=>61, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Development_and_Validation_of_Burkholderia_pseudomallei_Specific_Real_Time_PCR_Assays_for_Clinical_Environmental_or_Forensic_Detection_Applications/124996", "title"=>"Development and Validation of <em>Burkholderia pseudomallei</em>-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-05-18 01:23:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/636881"], "description"=>"<p>Green, the <i>B. pseudomallei</i>-specific TaqMan probe preferentially amplifies <i>B. pseudomallei</i> template; the non-<i>B.pseudomallei</i> TaqMan probe (red) amplifies well in <i>B. thailandensis-</i>like species and weakly in <i>B. thailandensis</i> and <i>B. oklahomensis</i>. Other <i>Burkholderia</i> spp. do not amplify.</p>", "links"=>[], "tags"=>["taqman", "dual", "probe"], "article_id"=>307338, "categories"=>["Cell Biology", "Microbiology", "Infectious Diseases", "Biotechnology", "Biological Sciences", "Genetics", "Evolutionary Biology"], "users"=>["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", "Bart J. Currie", "David M. Wagner", "Paul Keim", "Talima Pearson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0037723.g002", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_266152_TaqMan_dual_probe_assay_/307338", "title"=>"266152 TaqMan dual probe assay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-18 02:02:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/636692"], "description"=>"<p>Red, the <i>B. pseudomallei-</i>specific TaqMan probe amplifies only <i>B. pseudomallei</i> template; the non-<i>B.pseudomallei</i> TaqMan probe (green) amplifies well with <i>B. thailandensis</i> and <i>B. thailandensis-</i>like species and weakly with <i>B. oklahomensis</i> templates but not <i>B. pseudomallei</i>. Other <i>Burkholderia</i> spp. do not amplify with either probe.</p>", "links"=>[], "tags"=>["taqman", "dual", "probe"], "article_id"=>307154, "categories"=>["Cell Biology", "Microbiology", "Infectious Diseases", "Biotechnology", "Biological Sciences", "Genetics", "Evolutionary Biology"], "users"=>["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", "Bart J. Currie", "David M. Wagner", "Paul Keim", "Talima Pearson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0037723.g001", "stats"=>{"downloads"=>0, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_122018_TaqMan_dual_probe_assay_/307154", "title"=>"122018 TaqMan dual probe assay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-18 01:59:14"}

PMC Usage Stats | Further Information

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Relative Metric

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