Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infections
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/71533 |
Resumo: | A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the <i>Candida Panel</i> for the identification of <i>Candida</i> species, and the <i>Filamentous Fungi Panel</i> for the identification of <i>Aspergillus</i> species and <i>Rhizopus</i><i>arrhizus</i>. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes. |
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Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infectionsmolecular diagnosisfungal infectionsmultiplex PCRCandida spAspergillus spRhizopus arrhizusCiências Naturais::Ciências BiológicasScience & TechnologyA new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the <i>Candida Panel</i> for the identification of <i>Candida</i> species, and the <i>Filamentous Fungi Panel</i> for the identification of <i>Aspergillus</i> species and <i>Rhizopus</i><i>arrhizus</i>. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145- FEDER-007569) funded by national funds through the FCT I.P., by the ERDF through the COMPETE2020, by the strategic programme UID/BIA/04050/2019 funded by national funds through the FCT I.P., and co-founded by FEDER through Programa Operacional Regional do Norte, in the scope of the project U. Norte Inova. Joana Carvalho-Pereira was funded by FCT/Ministério da Educação e Ciência, grant number SFRH/BD/113384/2015. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors.Multidisciplinary Digital Publishing Institute (MDPI)Universidade do MinhoCarvalho-Pereira, JoanaFernandes, FilipaAraújo, RicardoSpringer, JanLoeffler, JuergenBuitrago, María JoséPais, CéliaSampaio, Paula20202020-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/71533engCarvalho-Pereira, J.; Fernandes, F.; Araújo, R.; Springer, J.; Loeffler, J.; Buitrago, M.J.; Pais, C.; Sampaio, P. Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections. J. Fungi 2020, 6, 308. https://doi.org/10.3390/jof60403082309-608X10.3390/jof6040308https://www.mdpi.com/2309-608X/6/4/308info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:22:47Zoai:repositorium.sdum.uminho.pt:1822/71533Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:16:20.769995Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infections |
title |
Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infections |
spellingShingle |
Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infections Carvalho-Pereira, Joana molecular diagnosis fungal infections multiplex PCR Candida sp Aspergillus sp Rhizopus arrhizus Ciências Naturais::Ciências Biológicas Science & Technology |
title_short |
Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infections |
title_full |
Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infections |
title_fullStr |
Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infections |
title_full_unstemmed |
Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infections |
title_sort |
Multiplex PCR based strategy for detection of fungal pathogen DNA in patients with suspected invasive fungal infections |
author |
Carvalho-Pereira, Joana |
author_facet |
Carvalho-Pereira, Joana Fernandes, Filipa Araújo, Ricardo Springer, Jan Loeffler, Juergen Buitrago, María José Pais, Célia Sampaio, Paula |
author_role |
author |
author2 |
Fernandes, Filipa Araújo, Ricardo Springer, Jan Loeffler, Juergen Buitrago, María José Pais, Célia Sampaio, Paula |
author2_role |
author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Carvalho-Pereira, Joana Fernandes, Filipa Araújo, Ricardo Springer, Jan Loeffler, Juergen Buitrago, María José Pais, Célia Sampaio, Paula |
dc.subject.por.fl_str_mv |
molecular diagnosis fungal infections multiplex PCR Candida sp Aspergillus sp Rhizopus arrhizus Ciências Naturais::Ciências Biológicas Science & Technology |
topic |
molecular diagnosis fungal infections multiplex PCR Candida sp Aspergillus sp Rhizopus arrhizus Ciências Naturais::Ciências Biológicas Science & Technology |
description |
A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the <i>Candida Panel</i> for the identification of <i>Candida</i> species, and the <i>Filamentous Fungi Panel</i> for the identification of <i>Aspergillus</i> species and <i>Rhizopus</i><i>arrhizus</i>. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020 2020-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/1822/71533 |
url |
http://hdl.handle.net/1822/71533 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Carvalho-Pereira, J.; Fernandes, F.; Araújo, R.; Springer, J.; Loeffler, J.; Buitrago, M.J.; Pais, C.; Sampaio, P. Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections. J. Fungi 2020, 6, 308. https://doi.org/10.3390/jof6040308 2309-608X 10.3390/jof6040308 https://www.mdpi.com/2309-608X/6/4/308 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Multidisciplinary Digital Publishing Institute (MDPI) |
publisher.none.fl_str_mv |
Multidisciplinary Digital Publishing Institute (MDPI) |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799132612175855616 |