Duplex realtime PCR method for Epstein-Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Brazilian Journal of Infectious Diseases |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1413-86702014000300271 |
Resumo: | 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 107-10 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. |
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Brazilian Journal of Infectious Diseases |
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Duplex realtime PCR method for Epstein-Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detectionEBVreal-time PCRviral loadPTLDINTRODUCTION: 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 107-10 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.Brazilian Society of Infectious Diseases2014-06-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1413-86702014000300271Brazilian Journal of Infectious Diseases v.18 n.3 2014reponame:Brazilian Journal of Infectious Diseasesinstname:Brazilian Society of Infectious Diseases (BSID)instacron:BSID10.1016/j.bjid.2013.07.011info:eu-repo/semantics/openAccessFellner,María DoloresDurand,KarinaRodriguez,MarceloIrazu,LucíaAlonio,VirginiaPicconi,María Alejandraeng2015-10-26T00:00:00Zoai:scielo:S1413-86702014000300271Revistahttps://www.bjid.org.br/https://old.scielo.br/oai/scielo-oai.phpbjid@bjid.org.br||lgoldani@ufrgs.br1678-43911413-8670opendoar:2015-10-26T00:00Brazilian Journal of Infectious Diseases - Brazilian Society of Infectious Diseases (BSID)false |
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 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 |
author |
Fellner,María Dolores |
author_facet |
Fellner,María Dolores Durand,Karina Rodriguez,Marcelo Irazu,Lucía Alonio,Virginia Picconi,María Alejandra |
author_role |
author |
author2 |
Durand,Karina Rodriguez,Marcelo Irazu,Lucía Alonio,Virginia Picconi,María Alejandra |
author2_role |
author author author author author |
dc.contributor.author.fl_str_mv |
Fellner,María Dolores Durand,Karina Rodriguez,Marcelo Irazu,Lucía Alonio,Virginia Picconi,María Alejandra |
dc.subject.por.fl_str_mv |
EBV real-time PCR viral load PTLD |
topic |
EBV real-time PCR viral load PTLD |
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 107-10 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 |
2014-06-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1413-86702014000300271 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1413-86702014000300271 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1016/j.bjid.2013.07.011 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Brazilian Society of Infectious Diseases |
publisher.none.fl_str_mv |
Brazilian Society of Infectious Diseases |
dc.source.none.fl_str_mv |
Brazilian Journal of Infectious Diseases v.18 n.3 2014 reponame:Brazilian Journal of Infectious Diseases instname:Brazilian Society of Infectious Diseases (BSID) instacron:BSID |
instname_str |
Brazilian Society of Infectious Diseases (BSID) |
instacron_str |
BSID |
institution |
BSID |
reponame_str |
Brazilian Journal of Infectious Diseases |
collection |
Brazilian Journal of Infectious Diseases |
repository.name.fl_str_mv |
Brazilian Journal of Infectious Diseases - Brazilian Society of Infectious Diseases (BSID) |
repository.mail.fl_str_mv |
bjid@bjid.org.br||lgoldani@ufrgs.br |
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1754209242873593856 |