Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection

Autores
Fellner, María Dolores; Durand, Karina; Rodríguez, Marcelo; Irazu, Lucía; Alonio, Lidia Virginia; Picconi, María Alejandra
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
INTRODUCTION: The quantification of circulating Epstein-Barr virus (EBV) DNA is used to monitor transplant patients as an early marker of Post-Transplant Lymphoproliferative Disorders (PTLD). So far no standardized methodology exists for such determination. OBJECTIVE: Our purpose was to develop and validate a real-time PCR assay to quantify EBV DNA in clinical samples from transplant recipients. METHODS: A duplex real-time PCR method was developed to amplify DNA from EBV and from a human gene. The EBV load was determined in peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal tissue from 64 non-transplanted patients with lymphoid-hypertrophy (Non-Tx), 47 transplant recipients without PTLD (Tx), 54 recipients with PTLD (Tx-PTLD), and 66 blood donors (BD). WinPEPI, version 11.14 software was used for statistical analysis. RESULTS: Analytical validation: the intra and inter-assays variation coefficients were less than 4.5% (EBV-reaction) and 3% (glyceraldehyde 3-phosphate dehydrogenase - GAPDH reaction). Linear ranges comprised 10(7)-10 EBV genome equivalents (gEq) (EBV-reaction) and 500,000-32 human gEq (GAPDH-reaction). The detection limit was 2.9 EBV gEq (EBV-reaction). Both reactions showed specificity. Application to clinical samples: higher levels of EBV were found in oropharyngeal tissue from transplanted groups with and without PTLD, compared to Non-Tx (p<0.05). The EBV load in PBMC from the groups of BD, Non-Tx, Tx and Tx-PTLD exhibited increasing levels (p<0.05). In BD, PBMC and plasma, EBV loads were undetectable. CONCLUSIONS: The performance of the assay was suitable for the required clinical application. The assay may be useful to monitor EBV infection in transplant patients, in particular in laboratories from low-income regions that cannot afford to use commercial assays.
Fil: Fellner, María Dolores. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Durand, Karina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Rodríguez, Marcelo. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Irazu, Lucía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Alonio, Lidia Virginia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Picconi, María Alejandra. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Materia
EBV
Real time PCR
Quantification
Transplant
Viral load
PTLD
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/33358
Acceder
id CONICETDig_0a1a8be9c5d45139a524be109e539611
oai_identifier_str oai:ri.conicet.gov.ar:11336/33358
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detectionFellner, María DoloresDurand, KarinaRodríguez, MarceloIrazu, LucíaAlonio, Lidia VirginiaPicconi, María AlejandraEBVReal time PCRQuantificationTransplantViral loadPTLDhttps://purl.org/becyt/ford/3.3https://purl.org/becyt/ford/3INTRODUCTION: The quantification of circulating Epstein-Barr virus (EBV) DNA is used to monitor transplant patients as an early marker of Post-Transplant Lymphoproliferative Disorders (PTLD). So far no standardized methodology exists for such determination. OBJECTIVE: Our purpose was to develop and validate a real-time PCR assay to quantify EBV DNA in clinical samples from transplant recipients. METHODS: A duplex real-time PCR method was developed to amplify DNA from EBV and from a human gene. The EBV load was determined in peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal tissue from 64 non-transplanted patients with lymphoid-hypertrophy (Non-Tx), 47 transplant recipients without PTLD (Tx), 54 recipients with PTLD (Tx-PTLD), and 66 blood donors (BD). WinPEPI, version 11.14 software was used for statistical analysis. RESULTS: Analytical validation: the intra and inter-assays variation coefficients were less than 4.5% (EBV-reaction) and 3% (glyceraldehyde 3-phosphate dehydrogenase - GAPDH reaction). Linear ranges comprised 10(7)-10 EBV genome equivalents (gEq) (EBV-reaction) and 500,000-32 human gEq (GAPDH-reaction). The detection limit was 2.9 EBV gEq (EBV-reaction). Both reactions showed specificity. Application to clinical samples: higher levels of EBV were found in oropharyngeal tissue from transplanted groups with and without PTLD, compared to Non-Tx (p<0.05). The EBV load in PBMC from the groups of BD, Non-Tx, Tx and Tx-PTLD exhibited increasing levels (p<0.05). In BD, PBMC and plasma, EBV loads were undetectable. CONCLUSIONS: The performance of the assay was suitable for the required clinical application. The assay may be useful to monitor EBV infection in transplant patients, in particular in laboratories from low-income regions that cannot afford to use commercial assays.Fil: Fellner, María Dolores. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; ArgentinaFil: Durand, Karina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; ArgentinaFil: Rodríguez, Marcelo. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; ArgentinaFil: Irazu, Lucía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; ArgentinaFil: Alonio, Lidia Virginia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Picconi, María Alejandra. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; ArgentinaElsevier2014-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/33358Durand, Karina; Rodríguez, Marcelo; Picconi, María Alejandra; Alonio, Lidia Virginia; Irazu, Lucía; Fellner, María Dolores; et al.; Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection; Elsevier; Brazilian Journal of Infectious Diseases; 18; 3; 1-2014; 271-2801413-86701678-4391CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://ref.scielo.org/h6fgk7info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S141386701300281Xinfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.bjid.2013.07.011info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:06:28Zoai:ri.conicet.gov.ar:11336/33358instacron: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:34982025-09-29 10:06:28.632CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
title Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
spellingShingle Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
Fellner, María Dolores
EBV
Real time PCR
Quantification
Transplant
Viral load
PTLD
title_short Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
title_full Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
title_fullStr Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
title_full_unstemmed Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
title_sort Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
dc.creator.none.fl_str_mv Fellner, María Dolores
Durand, Karina
Rodríguez, Marcelo
Irazu, Lucía
Alonio, Lidia Virginia
Picconi, María Alejandra
author Fellner, María Dolores
author_facet Fellner, María Dolores
Durand, Karina
Rodríguez, Marcelo
Irazu, Lucía
Alonio, Lidia Virginia
Picconi, María Alejandra
author_role author
author2 Durand, Karina
Rodríguez, Marcelo
Irazu, Lucía
Alonio, Lidia Virginia
Picconi, María Alejandra
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv EBV
Real time PCR
Quantification
Transplant
Viral load
PTLD
topic EBV
Real time PCR
Quantification
Transplant
Viral load
PTLD
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.3
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv INTRODUCTION: The quantification of circulating Epstein-Barr virus (EBV) DNA is used to monitor transplant patients as an early marker of Post-Transplant Lymphoproliferative Disorders (PTLD). So far no standardized methodology exists for such determination. OBJECTIVE: Our purpose was to develop and validate a real-time PCR assay to quantify EBV DNA in clinical samples from transplant recipients. METHODS: A duplex real-time PCR method was developed to amplify DNA from EBV and from a human gene. The EBV load was determined in peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal tissue from 64 non-transplanted patients with lymphoid-hypertrophy (Non-Tx), 47 transplant recipients without PTLD (Tx), 54 recipients with PTLD (Tx-PTLD), and 66 blood donors (BD). WinPEPI, version 11.14 software was used for statistical analysis. RESULTS: Analytical validation: the intra and inter-assays variation coefficients were less than 4.5% (EBV-reaction) and 3% (glyceraldehyde 3-phosphate dehydrogenase - GAPDH reaction). Linear ranges comprised 10(7)-10 EBV genome equivalents (gEq) (EBV-reaction) and 500,000-32 human gEq (GAPDH-reaction). The detection limit was 2.9 EBV gEq (EBV-reaction). Both reactions showed specificity. Application to clinical samples: higher levels of EBV were found in oropharyngeal tissue from transplanted groups with and without PTLD, compared to Non-Tx (p<0.05). The EBV load in PBMC from the groups of BD, Non-Tx, Tx and Tx-PTLD exhibited increasing levels (p<0.05). In BD, PBMC and plasma, EBV loads were undetectable. CONCLUSIONS: The performance of the assay was suitable for the required clinical application. The assay may be useful to monitor EBV infection in transplant patients, in particular in laboratories from low-income regions that cannot afford to use commercial assays.
Fil: Fellner, María Dolores. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Durand, Karina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Rodríguez, Marcelo. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Irazu, Lucía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
Fil: Alonio, Lidia Virginia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Picconi, María Alejandra. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina
description INTRODUCTION: The quantification of circulating Epstein-Barr virus (EBV) DNA is used to monitor transplant patients as an early marker of Post-Transplant Lymphoproliferative Disorders (PTLD). So far no standardized methodology exists for such determination. OBJECTIVE: Our purpose was to develop and validate a real-time PCR assay to quantify EBV DNA in clinical samples from transplant recipients. METHODS: A duplex real-time PCR method was developed to amplify DNA from EBV and from a human gene. The EBV load was determined in peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal tissue from 64 non-transplanted patients with lymphoid-hypertrophy (Non-Tx), 47 transplant recipients without PTLD (Tx), 54 recipients with PTLD (Tx-PTLD), and 66 blood donors (BD). WinPEPI, version 11.14 software was used for statistical analysis. RESULTS: Analytical validation: the intra and inter-assays variation coefficients were less than 4.5% (EBV-reaction) and 3% (glyceraldehyde 3-phosphate dehydrogenase - GAPDH reaction). Linear ranges comprised 10(7)-10 EBV genome equivalents (gEq) (EBV-reaction) and 500,000-32 human gEq (GAPDH-reaction). The detection limit was 2.9 EBV gEq (EBV-reaction). Both reactions showed specificity. Application to clinical samples: higher levels of EBV were found in oropharyngeal tissue from transplanted groups with and without PTLD, compared to Non-Tx (p<0.05). The EBV load in PBMC from the groups of BD, Non-Tx, Tx and Tx-PTLD exhibited increasing levels (p<0.05). In BD, PBMC and plasma, EBV loads were undetectable. CONCLUSIONS: The performance of the assay was suitable for the required clinical application. The assay may be useful to monitor EBV infection in transplant patients, in particular in laboratories from low-income regions that cannot afford to use commercial assays.
publishDate 2014
dc.date.none.fl_str_mv 2014-01
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/11336/33358
Durand, Karina; Rodríguez, Marcelo; Picconi, María Alejandra; Alonio, Lidia Virginia; Irazu, Lucía; Fellner, María Dolores; et al.; Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection; Elsevier; Brazilian Journal of Infectious Diseases; 18; 3; 1-2014; 271-280
1413-8670
1678-4391
CONICET Digital
CONICET
url http://hdl.handle.net/11336/33358
identifier_str_mv Durand, Karina; Rodríguez, Marcelo; Picconi, María Alejandra; Alonio, Lidia Virginia; Irazu, Lucía; Fellner, María Dolores; et al.; Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection; Elsevier; Brazilian Journal of Infectious Diseases; 18; 3; 1-2014; 271-280
1413-8670
1678-4391
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://ref.scielo.org/h6fgk7
info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S141386701300281X
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bjid.2013.07.011
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
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 13.070432

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