A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater

Scuticociliatosis is a serious disease that affects flatfish during culture and against which no effective control measures have yet been developed. Monitoring parasite levels in the water may be a valuable way of establishing the risk of infection and enabling appropriate control measures to be tak...

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Detalhes bibliográficos
Autores: Sueiro, Rosa-Ana, Lamas, Jesús, Palenzuela, Oswaldo, Gulias, Paola, Díez-Vives, Cristina, García-Ulloa, Alba, Leiro, José-Manuel
Tipo de documento: artigo
Estado:Versão publicada
Data de publicação:2022
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositório:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/284627
Acesso em linha:http://hdl.handle.net/10261/284627
Access Level:Acceso aberto
Palavra-chave:Scuticociliatosis
Philasterides dicentrarchi
Miamiensis avidus
Real-time quantitative
PCR
Internal transcribed spacer 2
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spelling A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawaterSueiro, Rosa-AnaLamas, JesúsPalenzuela, OswaldoGulias, PaolaDíez-Vives, CristinaGarcía-Ulloa, AlbaLeiro, José-ManuelScuticociliatosisPhilasterides dicentrarchiMiamiensis avidusReal-time quantitativePCRInternal transcribed spacer 2Scuticociliatosis is a serious disease that affects flatfish during culture and against which no effective control measures have yet been developed. Monitoring parasite levels in the water may be a valuable way of establishing the risk of infection and enabling appropriate control measures to be taken, thus representing an advance in controlling the disease. To achieve this objective, we have designed a quantitative real-time PCR (qPCR) assay using primers (f / r ITS2) and a hydrolysis probe that specifically amplify a region of the internal transcribed spacer 2 (ITS2) of the main aetiological agents of scuticociliatosis: Philasterides dicentrarchi and Miamiensis avidus. The slope (m), efficiency (E) and linearity (R2) determined from the standard curves generated are within the optimal values for qPCR. The high analytical sensitivity of the qPCR assay enables quantification of less than 120 pg of DNA per μL of reaction and detection of 1 ciliate per assay. The qPCR assay also exhibits high precision, with intra- and inter-assay coefficients of variation (CV) of respectively 0.27 and 7.57%. The protocol developed for isolating and quantifying ciliates seawater samples it has a recovery efficiency greater than 75% when the ciliate levels are between 103 and 2 × 103 ciliates/L and the turbidity of the water does not exceed one nephelometric turbidity unit (NTU). The real-time qPCR assay developed is a useful and appropriate tool for the specific and sensitive monitoring of scuticociliates in the water used in flatfish farms, enabling the establishment of effective prevention and control programmes.This study was financially supported by grants PID 2020-113087RB- I00 awarded by the Ministerio de Ciencia e Innovación (Spain) and the European Regional Development Fund (ERDF) (European Union), IDI-20200702 by Centro para el Desarrollo Tecnológico Industrial (CDTI) of the Ministerio de Ciencia e Innovación (Spain) and ED431C2021/26 from the Xunta de Galicia (Spain), and by the PARAFISHCONTROL project, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 634429.ElsevierMinisterio de Ciencia, Innovación y Universidades (España)Agencia Estatal de Investigación (España)Centro para el Desarrollo Tecnológico Industrial (España)Xunta de GaliciaEuropean CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2022202220222022info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/284627reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-113087RB-I00info:eu-repo/grantAgreement/EC/H2020/634429http://dx.doi.org/10.1016/j.aquaculture.2022.738303Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2846272026-05-22T06:33:51Z
dc.title.none.fl_str_mv A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater
title A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater
spellingShingle A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater
Sueiro, Rosa-Ana
Scuticociliatosis
Philasterides dicentrarchi
Miamiensis avidus
Real-time quantitative
PCR
Internal transcribed spacer 2
title_short A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater
title_full A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater
title_fullStr A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater
title_full_unstemmed A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater
title_sort A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater
dc.creator.none.fl_str_mv Sueiro, Rosa-Ana
Lamas, Jesús
Palenzuela, Oswaldo
Gulias, Paola
Díez-Vives, Cristina
García-Ulloa, Alba
Leiro, José-Manuel
author Sueiro, Rosa-Ana
author_facet Sueiro, Rosa-Ana
Lamas, Jesús
Palenzuela, Oswaldo
Gulias, Paola
Díez-Vives, Cristina
García-Ulloa, Alba
Leiro, José-Manuel
author_role author
author2 Lamas, Jesús
Palenzuela, Oswaldo
Gulias, Paola
Díez-Vives, Cristina
García-Ulloa, Alba
Leiro, José-Manuel
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia, Innovación y Universidades (España)
Agencia Estatal de Investigación (España)
Centro para el Desarrollo Tecnológico Industrial (España)
Xunta de Galicia
European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Scuticociliatosis
Philasterides dicentrarchi
Miamiensis avidus
Real-time quantitative
PCR
Internal transcribed spacer 2
topic Scuticociliatosis
Philasterides dicentrarchi
Miamiensis avidus
Real-time quantitative
PCR
Internal transcribed spacer 2
description Scuticociliatosis is a serious disease that affects flatfish during culture and against which no effective control measures have yet been developed. Monitoring parasite levels in the water may be a valuable way of establishing the risk of infection and enabling appropriate control measures to be taken, thus representing an advance in controlling the disease. To achieve this objective, we have designed a quantitative real-time PCR (qPCR) assay using primers (f / r ITS2) and a hydrolysis probe that specifically amplify a region of the internal transcribed spacer 2 (ITS2) of the main aetiological agents of scuticociliatosis: Philasterides dicentrarchi and Miamiensis avidus. The slope (m), efficiency (E) and linearity (R2) determined from the standard curves generated are within the optimal values for qPCR. The high analytical sensitivity of the qPCR assay enables quantification of less than 120 pg of DNA per μL of reaction and detection of 1 ciliate per assay. The qPCR assay also exhibits high precision, with intra- and inter-assay coefficients of variation (CV) of respectively 0.27 and 7.57%. The protocol developed for isolating and quantifying ciliates seawater samples it has a recovery efficiency greater than 75% when the ciliate levels are between 103 and 2 × 103 ciliates/L and the turbidity of the water does not exceed one nephelometric turbidity unit (NTU). The real-time qPCR assay developed is a useful and appropriate tool for the specific and sensitive monitoring of scuticociliates in the water used in flatfish farms, enabling the establishment of effective prevention and control programmes.
publishDate 2022
dc.date.none.fl_str_mv 2022
2022
2022
2022
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/284627
url http://hdl.handle.net/10261/284627
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-113087RB-I00
info:eu-repo/grantAgreement/EC/H2020/634429
http://dx.doi.org/10.1016/j.aquaculture.2022.738303
Sí
dc.rights.none.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 Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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