Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens

Detalhes bibliográficos
Autor(a) principal: Quemelo,Paulo Roberto Veiga
Data de Publicação: 2009
Outros Autores: Fonseca,Benedito Antônio Lopes da, Lima,Danielle Malta, Peres,Luiz Cesar
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Jornal Brasileiro de Patologia e Medicina Laboratorial (Online)
Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442009000200006
Resumo: The polymerase chain reaction (PCR) has provided diagnosis of archival material, but some fixation methods such as formalin damage DNA and, subsequently, affect PCR analysis, particularly paraffin-embedded tissues. PCR is known due to its high specificity and sensitivity, although some difficulties arise when formalinfixed and paraffin-embedded tissue is used. Not only does this occur due to protein cross-linking, which increases with longer fixation time, but it also happens due to the direct damage that formalin causes in the DNA itself. PCR was used to analyze placenta and fetal organs from 34 samples with suspected Parvovirus B19 infection. It was not possible to amplify Parvovirus B19 DNA using nested-PCR, probably due to the size of the amplicon generated with the first set of primers. We approached this problem by using only the second set of primers. Two out of 34 tissue samples (5,9%) were positive by PCR. However, PCR performed on corresponding fetal organs was negative in one of the two. We also observed a negative relation between the thickness of the tissue fragment and the positivity of the samples. In conclusion, although PCR is highly specific and sensitive in fresh or ideally fixed material, a careful standardization of PCR assays is necessary when using formalin fixed paraffin-embedded tissues by applying primers that require smaller DNA fragments for amplification.
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spelling Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimensPolymerase chain reactionParvovirus B19AmplificationThe polymerase chain reaction (PCR) has provided diagnosis of archival material, but some fixation methods such as formalin damage DNA and, subsequently, affect PCR analysis, particularly paraffin-embedded tissues. PCR is known due to its high specificity and sensitivity, although some difficulties arise when formalinfixed and paraffin-embedded tissue is used. Not only does this occur due to protein cross-linking, which increases with longer fixation time, but it also happens due to the direct damage that formalin causes in the DNA itself. PCR was used to analyze placenta and fetal organs from 34 samples with suspected Parvovirus B19 infection. It was not possible to amplify Parvovirus B19 DNA using nested-PCR, probably due to the size of the amplicon generated with the first set of primers. We approached this problem by using only the second set of primers. Two out of 34 tissue samples (5,9%) were positive by PCR. However, PCR performed on corresponding fetal organs was negative in one of the two. We also observed a negative relation between the thickness of the tissue fragment and the positivity of the samples. In conclusion, although PCR is highly specific and sensitive in fresh or ideally fixed material, a careful standardization of PCR assays is necessary when using formalin fixed paraffin-embedded tissues by applying primers that require smaller DNA fragments for amplification.Sociedade Brasileira de Patologia Clínica2009-04-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442009000200006Jornal Brasileiro de Patologia e Medicina Laboratorial v.45 n.2 2009reponame:Jornal Brasileiro de Patologia e Medicina Laboratorial (Online)instname:Sociedade Brasileira de Patologia (SBP)instacron:SBP10.1590/S1676-24442009000200006info:eu-repo/semantics/openAccessQuemelo,Paulo Roberto VeigaFonseca,Benedito Antônio Lopes daLima,Danielle MaltaPeres,Luiz Cesareng2009-08-04T00:00:00Zoai:scielo:S1676-24442009000200006Revistahttp://www.scielo.br/jbpmlhttps://old.scielo.br/oai/scielo-oai.php||jbpml@sbpc.org.br1678-47741676-2444opendoar:2009-08-04T00:00Jornal Brasileiro de Patologia e Medicina Laboratorial (Online) - Sociedade Brasileira de Patologia (SBP)false
dc.title.none.fl_str_mv Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens
title Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens
spellingShingle Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens
Quemelo,Paulo Roberto Veiga
Polymerase chain reaction
Parvovirus B19
Amplification
title_short Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens
title_full Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens
title_fullStr Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens
title_full_unstemmed Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens
title_sort Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens
author Quemelo,Paulo Roberto Veiga
author_facet Quemelo,Paulo Roberto Veiga
Fonseca,Benedito Antônio Lopes da
Lima,Danielle Malta
Peres,Luiz Cesar
author_role author
author2 Fonseca,Benedito Antônio Lopes da
Lima,Danielle Malta
Peres,Luiz Cesar
author2_role author
author
author
dc.contributor.author.fl_str_mv Quemelo,Paulo Roberto Veiga
Fonseca,Benedito Antônio Lopes da
Lima,Danielle Malta
Peres,Luiz Cesar
dc.subject.por.fl_str_mv Polymerase chain reaction
Parvovirus B19
Amplification
topic Polymerase chain reaction
Parvovirus B19
Amplification
description The polymerase chain reaction (PCR) has provided diagnosis of archival material, but some fixation methods such as formalin damage DNA and, subsequently, affect PCR analysis, particularly paraffin-embedded tissues. PCR is known due to its high specificity and sensitivity, although some difficulties arise when formalinfixed and paraffin-embedded tissue is used. Not only does this occur due to protein cross-linking, which increases with longer fixation time, but it also happens due to the direct damage that formalin causes in the DNA itself. PCR was used to analyze placenta and fetal organs from 34 samples with suspected Parvovirus B19 infection. It was not possible to amplify Parvovirus B19 DNA using nested-PCR, probably due to the size of the amplicon generated with the first set of primers. We approached this problem by using only the second set of primers. Two out of 34 tissue samples (5,9%) were positive by PCR. However, PCR performed on corresponding fetal organs was negative in one of the two. We also observed a negative relation between the thickness of the tissue fragment and the positivity of the samples. In conclusion, although PCR is highly specific and sensitive in fresh or ideally fixed material, a careful standardization of PCR assays is necessary when using formalin fixed paraffin-embedded tissues by applying primers that require smaller DNA fragments for amplification.
publishDate 2009
dc.date.none.fl_str_mv 2009-04-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
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dc.identifier.uri.fl_str_mv http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442009000200006
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442009000200006
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/S1676-24442009000200006
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dc.publisher.none.fl_str_mv
Sociedade Brasileira de Patologia Clínica
publisher.none.fl_str_mv
Sociedade Brasileira de Patologia Clínica
dc.source.none.fl_str_mv Jornal Brasileiro de Patologia e Medicina Laboratorial v.45 n.2 2009
reponame:Jornal Brasileiro de Patologia e Medicina Laboratorial (Online)
instname:Sociedade Brasileira de Patologia (SBP)
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reponame_str Jornal Brasileiro de Patologia e Medicina Laboratorial (Online)
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