A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes

Detalhes bibliográficos
Autor(a) principal: Sitnik, Roberta
Data de Publicação: 2010
Outros Autores: Paes, Ângela, Mangueira, Cristovão Pitangueira, Pinho, João Renato Rebello
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Revista do Instituto de Medicina Tropical de São Paulo
Texto Completo: https://www.revistas.usp.br/rimtsp/article/view/31317
Resumo: Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. Besides genotype, quantitative analysis of HBV infection is extensively used for monitoring disease progression and treatment. Affordable viral load monitoring is desirable in resource-limited settings and it has been already shown to be useful in developing countries for other viruses such as Hepatitis C virus (HCV) and HIV. In this paper, we describe the validation of a real-time PCR assay for HBV DNA quantification with TaqMan chemistry and MGB probes. Primers and probes were designed using an alignment of sequences from all HBV genotypes in order to equally amplify all of them. The assay is internally controlled and was standardized with an international HBV panel. Its efficacy was evaluated comparing the results with two other methods: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) and another real-time PCR from a reference laboratory. Intra-assay and inter-assay reproducibilities were determined and the mean of CV values obtained were 0.12 and 0.09, respectively. The assay was validated with a broad dynamic range and is efficient for amplifying all HBV genotypes, providing a good option to quantify HBV DNA as a routine procedure, with a cheap and reliable protocol.
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spelling A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes Ensaio quantitativo em tempo real para o DNA do vírus da hepatite B (HBV) desenvolvido para detectar todos os genótipos do HBV HBV DNA quantificationReal Time PCRHepatitis B virusViral load Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. Besides genotype, quantitative analysis of HBV infection is extensively used for monitoring disease progression and treatment. Affordable viral load monitoring is desirable in resource-limited settings and it has been already shown to be useful in developing countries for other viruses such as Hepatitis C virus (HCV) and HIV. In this paper, we describe the validation of a real-time PCR assay for HBV DNA quantification with TaqMan chemistry and MGB probes. Primers and probes were designed using an alignment of sequences from all HBV genotypes in order to equally amplify all of them. The assay is internally controlled and was standardized with an international HBV panel. Its efficacy was evaluated comparing the results with two other methods: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) and another real-time PCR from a reference laboratory. Intra-assay and inter-assay reproducibilities were determined and the mean of CV values obtained were 0.12 and 0.09, respectively. The assay was validated with a broad dynamic range and is efficient for amplifying all HBV genotypes, providing a good option to quantify HBV DNA as a routine procedure, with a cheap and reliable protocol. O vírus da Hepatite B (HBV) é uma das principais causas de doença crônica do fígado no mundo. Além do genótipo, a análise quantitativa do HBV é amplamente utilizada para monitorar a progressão da doença e o tratamento. Em locais com recursos escassos, métodos baratos para o monitoramento da carga viral são desejáveis e, em países em desenvolvimento, sua utilidade já foi demonstrada para outros vírus, como o da Hepatite C e HIV. Neste trabalho, descrevemos a validação de um teste de PCR em Tempo Real para a quantificação do DNA do HBV utilizando sondas Taqman/MGB. Os oligos e sondas foram escolhidos usando um alinhamento contendo seqüências de todos os genótipos do HBV para garantir uma amplificação igual de todos eles. O teste possui um controle interno e foi padronizado com um painel internacional de HBV. Sua eficácia foi testada comparando-se os resultados com outros dois métodos: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) e outro PCR em tempo real realizado em um laboratório de referência. As reprodutibilidades intra e inter-ensaio foram determinadas e a média dos valores de CV obtidos foram de 0,12 e 0,09, respectivamente. O teste foi validado com uma ampla faixa dinâmica e amplificou com eficiência os diferentes genótipos de HBV, fornecendo uma boa opção para a quantificação de rotina do DNA do HBV, com um protocolo barato e confiável. Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo2010-06-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://www.revistas.usp.br/rimtsp/article/view/31317Revista do Instituto de Medicina Tropical de São Paulo; Vol. 52 No. 3 (2010); 119-124 Revista do Instituto de Medicina Tropical de São Paulo; Vol. 52 Núm. 3 (2010); 119-124 Revista do Instituto de Medicina Tropical de São Paulo; v. 52 n. 3 (2010); 119-124 1678-99460036-4665reponame:Revista do Instituto de Medicina Tropical de São Pauloinstname:Instituto de Medicina Tropical (IMT)instacron:IMTenghttps://www.revistas.usp.br/rimtsp/article/view/31317/33202Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Pauloinfo:eu-repo/semantics/openAccessSitnik, RobertaPaes, ÂngelaMangueira, Cristovão PitangueiraPinho, João Renato Rebello2012-07-07T19:30:54Zoai:revistas.usp.br:article/31317Revistahttp://www.revistas.usp.br/rimtsp/indexPUBhttps://www.revistas.usp.br/rimtsp/oai||revimtsp@usp.br1678-99460036-4665opendoar:2022-12-13T16:51:59.623478Revista do Instituto de Medicina Tropical de São Paulo - Instituto de Medicina Tropical (IMT)true
dc.title.none.fl_str_mv A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes
Ensaio quantitativo em tempo real para o DNA do vírus da hepatite B (HBV) desenvolvido para detectar todos os genótipos do HBV
title A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes
spellingShingle A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes
Sitnik, Roberta
HBV DNA quantification
Real Time PCR
Hepatitis B virus
Viral load
title_short A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes
title_full A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes
title_fullStr A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes
title_full_unstemmed A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes
title_sort A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes
author Sitnik, Roberta
author_facet Sitnik, Roberta
Paes, Ângela
Mangueira, Cristovão Pitangueira
Pinho, João Renato Rebello
author_role author
author2 Paes, Ângela
Mangueira, Cristovão Pitangueira
Pinho, João Renato Rebello
author2_role author
author
author
dc.contributor.author.fl_str_mv Sitnik, Roberta
Paes, Ângela
Mangueira, Cristovão Pitangueira
Pinho, João Renato Rebello
dc.subject.por.fl_str_mv HBV DNA quantification
Real Time PCR
Hepatitis B virus
Viral load
topic HBV DNA quantification
Real Time PCR
Hepatitis B virus
Viral load
description Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. Besides genotype, quantitative analysis of HBV infection is extensively used for monitoring disease progression and treatment. Affordable viral load monitoring is desirable in resource-limited settings and it has been already shown to be useful in developing countries for other viruses such as Hepatitis C virus (HCV) and HIV. In this paper, we describe the validation of a real-time PCR assay for HBV DNA quantification with TaqMan chemistry and MGB probes. Primers and probes were designed using an alignment of sequences from all HBV genotypes in order to equally amplify all of them. The assay is internally controlled and was standardized with an international HBV panel. Its efficacy was evaluated comparing the results with two other methods: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) and another real-time PCR from a reference laboratory. Intra-assay and inter-assay reproducibilities were determined and the mean of CV values obtained were 0.12 and 0.09, respectively. The assay was validated with a broad dynamic range and is efficient for amplifying all HBV genotypes, providing a good option to quantify HBV DNA as a routine procedure, with a cheap and reliable protocol.
publishDate 2010
dc.date.none.fl_str_mv 2010-06-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://www.revistas.usp.br/rimtsp/article/view/31317
url https://www.revistas.usp.br/rimtsp/article/view/31317
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv https://www.revistas.usp.br/rimtsp/article/view/31317/33202
dc.rights.driver.fl_str_mv Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Paulo
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Paulo
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo
publisher.none.fl_str_mv Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo
dc.source.none.fl_str_mv Revista do Instituto de Medicina Tropical de São Paulo; Vol. 52 No. 3 (2010); 119-124
Revista do Instituto de Medicina Tropical de São Paulo; Vol. 52 Núm. 3 (2010); 119-124
Revista do Instituto de Medicina Tropical de São Paulo; v. 52 n. 3 (2010); 119-124
1678-9946
0036-4665
reponame:Revista do Instituto de Medicina Tropical de São Paulo
instname:Instituto de Medicina Tropical (IMT)
instacron:IMT
instname_str Instituto de Medicina Tropical (IMT)
instacron_str IMT
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reponame_str Revista do Instituto de Medicina Tropical de São Paulo
collection Revista do Instituto de Medicina Tropical de São Paulo
repository.name.fl_str_mv Revista do Instituto de Medicina Tropical de São Paulo - Instituto de Medicina Tropical (IMT)
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