Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus
Autor(a) principal: | |
---|---|
Data de Publicação: | 2022 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | https://hdl.handle.net/1822/80193 |
Resumo: | Introduction Multiplex quantitative polymerase chain reaction (qPCR) methods for the detection of Aspergillus spp. based only on SYBR Green and melting curve analysis of PCR products are difficult to develop because most targets are located within ITS regions. The aim of this study was to adapt our previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis to provide a qPCR method. Methods A SYBR Green-based real-time PCR assay was optimized to detect A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus in a multiplex assay and applied to cultured fungi and spiked plasma. Results Different melting temperatures allowed identification of all five pathogens and discrimination between them, even in samples with low amounts of fungal gDNA (from 1.3 to 33.0 pg/mu L), which has been reported previously as problematic. No false-positive results were obtained for non-target species, including bacteria and human DNA. This method allowed detection of fungal pathogens in human plasma spiked with fungal DNA and in coinfections of A. niger/R. arrhizus. Discussion This work provides evidence for the use of a qPCR multiplex method based on SYBR Green and melting curve analysis of PCR products for the detection of A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus. The proposed method is simpler and less expensive than available kits based on fluorescent probes and can be used for aiding diagnosis of the most relevant invasive filamentous fungi, particularly in low-income health care institutions. |
id |
RCAP_1266f11c7511b59b8beb52ee6d75d66f |
---|---|
oai_identifier_str |
oai:repositorium.sdum.uminho.pt:1822/80193 |
network_acronym_str |
RCAP |
network_name_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository_id_str |
7160 |
spelling |
Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizusCiências Naturais::Ciências BiológicasScience & TechnologyIntroduction Multiplex quantitative polymerase chain reaction (qPCR) methods for the detection of Aspergillus spp. based only on SYBR Green and melting curve analysis of PCR products are difficult to develop because most targets are located within ITS regions. The aim of this study was to adapt our previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis to provide a qPCR method. Methods A SYBR Green-based real-time PCR assay was optimized to detect A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus in a multiplex assay and applied to cultured fungi and spiked plasma. Results Different melting temperatures allowed identification of all five pathogens and discrimination between them, even in samples with low amounts of fungal gDNA (from 1.3 to 33.0 pg/mu L), which has been reported previously as problematic. No false-positive results were obtained for non-target species, including bacteria and human DNA. This method allowed detection of fungal pathogens in human plasma spiked with fungal DNA and in coinfections of A. niger/R. arrhizus. Discussion This work provides evidence for the use of a qPCR multiplex method based on SYBR Green and melting curve analysis of PCR products for the detection of A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus. The proposed method is simpler and less expensive than available kits based on fluorescent probes and can be used for aiding diagnosis of the most relevant invasive filamentous fungi, particularly in low-income health care institutions.(undefined)SpringerUniversidade do MinhoMendonca, AlexandreCarvalho-Pereira, JoanaFranco-Duarte, RicardoSampaio, Paula2022-06-162022-06-16T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/1822/80193engMendonça, A., Carvalho-Pereira, J., Franco-Duarte, R. et al. Optimization of a Quantitative PCR Methodology for Detection of Aspergillus spp. and Rhizopus arrhizus. Mol Diagn Ther 26, 511–525 (2022). https://doi.org/10.1007/s40291-022-00595-11177-10621179-200010.1007/s40291-022-00595-135710958https://link.springer.com/article/10.1007/s40291-022-00595-1info: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:RCAAP2023-07-21T12:35:33Zoai:repositorium.sdum.uminho.pt:1822/80193Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:31:25.062354Repositório 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 |
Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus |
title |
Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus |
spellingShingle |
Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus Mendonca, Alexandre Ciências Naturais::Ciências Biológicas Science & Technology |
title_short |
Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus |
title_full |
Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus |
title_fullStr |
Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus |
title_full_unstemmed |
Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus |
title_sort |
Optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus |
author |
Mendonca, Alexandre |
author_facet |
Mendonca, Alexandre Carvalho-Pereira, Joana Franco-Duarte, Ricardo Sampaio, Paula |
author_role |
author |
author2 |
Carvalho-Pereira, Joana Franco-Duarte, Ricardo Sampaio, Paula |
author2_role |
author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Mendonca, Alexandre Carvalho-Pereira, Joana Franco-Duarte, Ricardo Sampaio, Paula |
dc.subject.por.fl_str_mv |
Ciências Naturais::Ciências Biológicas Science & Technology |
topic |
Ciências Naturais::Ciências Biológicas Science & Technology |
description |
Introduction Multiplex quantitative polymerase chain reaction (qPCR) methods for the detection of Aspergillus spp. based only on SYBR Green and melting curve analysis of PCR products are difficult to develop because most targets are located within ITS regions. The aim of this study was to adapt our previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis to provide a qPCR method. Methods A SYBR Green-based real-time PCR assay was optimized to detect A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus in a multiplex assay and applied to cultured fungi and spiked plasma. Results Different melting temperatures allowed identification of all five pathogens and discrimination between them, even in samples with low amounts of fungal gDNA (from 1.3 to 33.0 pg/mu L), which has been reported previously as problematic. No false-positive results were obtained for non-target species, including bacteria and human DNA. This method allowed detection of fungal pathogens in human plasma spiked with fungal DNA and in coinfections of A. niger/R. arrhizus. Discussion This work provides evidence for the use of a qPCR multiplex method based on SYBR Green and melting curve analysis of PCR products for the detection of A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus. The proposed method is simpler and less expensive than available kits based on fluorescent probes and can be used for aiding diagnosis of the most relevant invasive filamentous fungi, particularly in low-income health care institutions. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-06-16 2022-06-16T00:00:00Z |
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 |
https://hdl.handle.net/1822/80193 |
url |
https://hdl.handle.net/1822/80193 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Mendonça, A., Carvalho-Pereira, J., Franco-Duarte, R. et al. Optimization of a Quantitative PCR Methodology for Detection of Aspergillus spp. and Rhizopus arrhizus. Mol Diagn Ther 26, 511–525 (2022). https://doi.org/10.1007/s40291-022-00595-1 1177-1062 1179-2000 10.1007/s40291-022-00595-1 35710958 https://link.springer.com/article/10.1007/s40291-022-00595-1 |
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 |
Springer |
publisher.none.fl_str_mv |
Springer |
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 |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
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 |
repository.mail.fl_str_mv |
|
_version_ |
1799132822908174336 |