Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto

Autores
Kulik, Tomasz; Ostrowska, Anna; Buśko, Maciej; Pasquali, Matias; Beyer, Marco; Stenglein, Sebastian Alberto; Załuski, Dariusz; Sawicki, Jakub; Treder, Kinga; Perkowski, Juliusz
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
An ascomycete fungus, Fusarium graminearum sensu stricto (s.s.), is the major cause of Fusarium head blight (FHB), a devastating disease of cereals worldwide. The fungus contaminates crops with mycotoxins, which pose a serious threat to food and feed safety. In this study, we developed a highly sensitive mitochondrial based qPCR assay (FgMito qPCR) for quantification of F. graminearum s.s. To ensure high sensitivity of the assay, primers and a Minor-groove binding (MGB) probe were designed based on multi-copy mitochondrial DNA. The FgMito assay was successfully validated against a range of geographically diverse F. graminearum s.s. strains to ensure uniformity of the assay at an intraspecific level, as well as with other fungal species to ensure specificity. The assay was further evaluated in terms of efficiency and sensitivity against a test panel of different F. graminearum s.s. strains with various levels of pure fungal DNA and in the presence of wheat background DNA. The results showed a high efficiency of the assay developed, ranging from 93% to 101% with r2-values of > 0.99. We further showed that three low concentrations of fungal template 2 pg, 0.6 pg and 0.2 pg could be reliably quantified in the presence of wheat background DNA. The FgMito assay was used to quantify F. graminearum s.s. DNA on 65 field samples from a range of hosts with defined levels of trichothecenes. We revealed a significant positive correlation between fungal DNA quantity and the sum of trichothecenes. Lastly, we showed a higher sensitivity of the FgMito assay than the nuclear based qPCR assay for F. graminearum s.s. by comparing Ct-values from both assays.
Fil: Kulik, Tomasz. University of Warmia and Mazury; Polonia
Fil: Ostrowska, Anna. Poznań University of Life Sciences. Department of Chemistry; Polonia
Fil: Buśko, Maciej. Poznań University of Life Sciences. Department of Chemistry; Polonia
Fil: Pasquali, Matias. Luxembourg Institute of Science and Technology; Luxemburgo
Fil: Beyer, Marco. Luxembourg Institute of Science and Technology; Luxemburgo
Fil: Stenglein, Sebastian Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnolológico Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología. Laboratorio de Biologia Funcional y Biotecnología; Argentina. Fundación para Investigaciones Biológicas Aplicadas; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires; Argentina
Fil: Załuski, Dariusz. University of Warmia and Mazury; Polonia
Fil: Sawicki, Jakub. University of Warmia and Mazury; Polonia
Fil: Treder, Kinga. University of Warmia and Mazury; Polonia
Fil: Perkowski, Juliusz. Poznań University of Life Sciences. Department of Chemistry; Polonia
Materia
Qpcr
Fusarium Graminearum Sensu Stricto
Quantification
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/27068
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/27068
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu strictoKulik, TomaszOstrowska, AnnaBuśko, MaciejPasquali, MatiasBeyer, MarcoStenglein, Sebastian AlbertoZałuski, DariuszSawicki, JakubTreder, KingaPerkowski, JuliuszQpcrFusarium Graminearum Sensu StrictoQuantificationhttps://purl.org/becyt/ford/4.1https://purl.org/becyt/ford/4https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1https://purl.org/becyt/ford/4.4https://purl.org/becyt/ford/4An ascomycete fungus, Fusarium graminearum sensu stricto (s.s.), is the major cause of Fusarium head blight (FHB), a devastating disease of cereals worldwide. The fungus contaminates crops with mycotoxins, which pose a serious threat to food and feed safety. In this study, we developed a highly sensitive mitochondrial based qPCR assay (FgMito qPCR) for quantification of F. graminearum s.s. To ensure high sensitivity of the assay, primers and a Minor-groove binding (MGB) probe were designed based on multi-copy mitochondrial DNA. The FgMito assay was successfully validated against a range of geographically diverse F. graminearum s.s. strains to ensure uniformity of the assay at an intraspecific level, as well as with other fungal species to ensure specificity. The assay was further evaluated in terms of efficiency and sensitivity against a test panel of different F. graminearum s.s. strains with various levels of pure fungal DNA and in the presence of wheat background DNA. The results showed a high efficiency of the assay developed, ranging from 93% to 101% with r2-values of > 0.99. We further showed that three low concentrations of fungal template 2 pg, 0.6 pg and 0.2 pg could be reliably quantified in the presence of wheat background DNA. The FgMito assay was used to quantify F. graminearum s.s. DNA on 65 field samples from a range of hosts with defined levels of trichothecenes. We revealed a significant positive correlation between fungal DNA quantity and the sum of trichothecenes. Lastly, we showed a higher sensitivity of the FgMito assay than the nuclear based qPCR assay for F. graminearum s.s. by comparing Ct-values from both assays.Fil: Kulik, Tomasz. University of Warmia and Mazury; PoloniaFil: Ostrowska, Anna. Poznań University of Life Sciences. Department of Chemistry; PoloniaFil: Buśko, Maciej. Poznań University of Life Sciences. Department of Chemistry; PoloniaFil: Pasquali, Matias. Luxembourg Institute of Science and Technology; LuxemburgoFil: Beyer, Marco. Luxembourg Institute of Science and Technology; LuxemburgoFil: Stenglein, Sebastian Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnolológico Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología. Laboratorio de Biologia Funcional y Biotecnología; Argentina. Fundación para Investigaciones Biológicas Aplicadas; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires; ArgentinaFil: Załuski, Dariusz. University of Warmia and Mazury; PoloniaFil: Sawicki, Jakub. University of Warmia and Mazury; PoloniaFil: Treder, Kinga. University of Warmia and Mazury; PoloniaFil: Perkowski, Juliusz. Poznań University of Life Sciences. Department of Chemistry; PoloniaElsevier Science2015-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/27068Kulik, Tomasz; Ostrowska, Anna; Buśko, Maciej; Pasquali, Matias; Beyer, Marco; et al.; Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto; Elsevier Science; International Journal of Food Microbiology; 210; 6-2015; 16-230168-1605CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.ijfoodmicro.2015.06.012info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0168160515300362info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T15:27:07Zoai:ri.conicet.gov.ar:11336/27068instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-15 15:27:07.924CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto
title Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto
spellingShingle Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto
Kulik, Tomasz
Qpcr
Fusarium Graminearum Sensu Stricto
Quantification
title_short Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto
title_full Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto
title_fullStr Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto
title_full_unstemmed Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto
title_sort Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto
dc.creator.none.fl_str_mv Kulik, Tomasz
Ostrowska, Anna
Buśko, Maciej
Pasquali, Matias
Beyer, Marco
Stenglein, Sebastian Alberto
Załuski, Dariusz
Sawicki, Jakub
Treder, Kinga
Perkowski, Juliusz
author Kulik, Tomasz
author_facet Kulik, Tomasz
Ostrowska, Anna
Buśko, Maciej
Pasquali, Matias
Beyer, Marco
Stenglein, Sebastian Alberto
Załuski, Dariusz
Sawicki, Jakub
Treder, Kinga
Perkowski, Juliusz
author_role author
author2 Ostrowska, Anna
Buśko, Maciej
Pasquali, Matias
Beyer, Marco
Stenglein, Sebastian Alberto
Załuski, Dariusz
Sawicki, Jakub
Treder, Kinga
Perkowski, Juliusz
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Qpcr
Fusarium Graminearum Sensu Stricto
Quantification
topic Qpcr
Fusarium Graminearum Sensu Stricto
Quantification
purl_subject.fl_str_mv https://purl.org/becyt/ford/4.1
https://purl.org/becyt/ford/4
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
https://purl.org/becyt/ford/4.4
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv An ascomycete fungus, Fusarium graminearum sensu stricto (s.s.), is the major cause of Fusarium head blight (FHB), a devastating disease of cereals worldwide. The fungus contaminates crops with mycotoxins, which pose a serious threat to food and feed safety. In this study, we developed a highly sensitive mitochondrial based qPCR assay (FgMito qPCR) for quantification of F. graminearum s.s. To ensure high sensitivity of the assay, primers and a Minor-groove binding (MGB) probe were designed based on multi-copy mitochondrial DNA. The FgMito assay was successfully validated against a range of geographically diverse F. graminearum s.s. strains to ensure uniformity of the assay at an intraspecific level, as well as with other fungal species to ensure specificity. The assay was further evaluated in terms of efficiency and sensitivity against a test panel of different F. graminearum s.s. strains with various levels of pure fungal DNA and in the presence of wheat background DNA. The results showed a high efficiency of the assay developed, ranging from 93% to 101% with r2-values of > 0.99. We further showed that three low concentrations of fungal template 2 pg, 0.6 pg and 0.2 pg could be reliably quantified in the presence of wheat background DNA. The FgMito assay was used to quantify F. graminearum s.s. DNA on 65 field samples from a range of hosts with defined levels of trichothecenes. We revealed a significant positive correlation between fungal DNA quantity and the sum of trichothecenes. Lastly, we showed a higher sensitivity of the FgMito assay than the nuclear based qPCR assay for F. graminearum s.s. by comparing Ct-values from both assays.
Fil: Kulik, Tomasz. University of Warmia and Mazury; Polonia
Fil: Ostrowska, Anna. Poznań University of Life Sciences. Department of Chemistry; Polonia
Fil: Buśko, Maciej. Poznań University of Life Sciences. Department of Chemistry; Polonia
Fil: Pasquali, Matias. Luxembourg Institute of Science and Technology; Luxemburgo
Fil: Beyer, Marco. Luxembourg Institute of Science and Technology; Luxemburgo
Fil: Stenglein, Sebastian Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnolológico Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología. Laboratorio de Biologia Funcional y Biotecnología; Argentina. Fundación para Investigaciones Biológicas Aplicadas; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires; Argentina
Fil: Załuski, Dariusz. University of Warmia and Mazury; Polonia
Fil: Sawicki, Jakub. University of Warmia and Mazury; Polonia
Fil: Treder, Kinga. University of Warmia and Mazury; Polonia
Fil: Perkowski, Juliusz. Poznań University of Life Sciences. Department of Chemistry; Polonia
description An ascomycete fungus, Fusarium graminearum sensu stricto (s.s.), is the major cause of Fusarium head blight (FHB), a devastating disease of cereals worldwide. The fungus contaminates crops with mycotoxins, which pose a serious threat to food and feed safety. In this study, we developed a highly sensitive mitochondrial based qPCR assay (FgMito qPCR) for quantification of F. graminearum s.s. To ensure high sensitivity of the assay, primers and a Minor-groove binding (MGB) probe were designed based on multi-copy mitochondrial DNA. The FgMito assay was successfully validated against a range of geographically diverse F. graminearum s.s. strains to ensure uniformity of the assay at an intraspecific level, as well as with other fungal species to ensure specificity. The assay was further evaluated in terms of efficiency and sensitivity against a test panel of different F. graminearum s.s. strains with various levels of pure fungal DNA and in the presence of wheat background DNA. The results showed a high efficiency of the assay developed, ranging from 93% to 101% with r2-values of > 0.99. We further showed that three low concentrations of fungal template 2 pg, 0.6 pg and 0.2 pg could be reliably quantified in the presence of wheat background DNA. The FgMito assay was used to quantify F. graminearum s.s. DNA on 65 field samples from a range of hosts with defined levels of trichothecenes. We revealed a significant positive correlation between fungal DNA quantity and the sum of trichothecenes. Lastly, we showed a higher sensitivity of the FgMito assay than the nuclear based qPCR assay for F. graminearum s.s. by comparing Ct-values from both assays.
publishDate 2015
dc.date.none.fl_str_mv 2015-06
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/27068
Kulik, Tomasz; Ostrowska, Anna; Buśko, Maciej; Pasquali, Matias; Beyer, Marco; et al.; Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto; Elsevier Science; International Journal of Food Microbiology; 210; 6-2015; 16-23
0168-1605
CONICET Digital
CONICET
url http://hdl.handle.net/11336/27068
identifier_str_mv Kulik, Tomasz; Ostrowska, Anna; Buśko, Maciej; Pasquali, Matias; Beyer, Marco; et al.; Development of an FgMito assay: a highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto; Elsevier Science; International Journal of Food Microbiology; 210; 6-2015; 16-23
0168-1605
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.ijfoodmicro.2015.06.012
info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0168160515300362
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.22299

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