Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting

Detalhes bibliográficos
Autor(a) principal: Ferreira, Beatriz Iandra da Silva
Data de Publicação: 2021
Outros Autores: Gomes, Natália Lins da Silva, Coelho, Wagner Luis da Costa Nunes Pimentel, Costa, Vanessa Duarte da, Carneiro, Vanessa Cristine de Souza, Kader, Rafael Lopes, Amaro, Marisa Pimentel, Villar, Lívia Melo, Miyajima, Fábio, Alves-Leon, Soniza Vieira, Paula, Vanessa Salete de, Leon, Luciane Almeida Amado, Moreira, Otacilio Cruz
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
Texto Completo: https://www.arca.fiocruz.br/handle/icict/50731
Resumo: Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Real Time PCR Platform RPT09A. Rio de Janeiro, RJ, Brasil.
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spelling Ferreira, Beatriz Iandra da SilvaGomes, Natália Lins da SilvaCoelho, Wagner Luis da Costa Nunes PimentelCosta, Vanessa Duarte daCarneiro, Vanessa Cristine de SouzaKader, Rafael LopesAmaro, Marisa PimentelVillar, Lívia MeloMiyajima, FábioAlves-Leon, Soniza VieiraPaula, Vanessa Salete deLeon, Luciane Almeida AmadoMoreira, Otacilio Cruz2022-01-11T18:21:22Z2022-01-11T18:21:22Z2021FERREIRA, Beatriz Iandra da Silva et al. Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting. Plos One, v. 16, n. 11, p. 1-17, Nov. 2021.1932-6203https://www.arca.fiocruz.br/handle/icict/5073110.1371/journal.pone.0260087engPublic Library of ScienceValidaçãoEnsaio molecularDiagnóstico de COVID-19Tempor realPCRFusão de alta resoluçãoValidationMolecular assayDiagnostic of COVID-19Real time PCRHigh resolution meltingValidation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution meltinginfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Real Time PCR Platform RPT09A. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Real Time PCR Platform RPT09A. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Fiocruz Ceará. Eusébio, CE, Brasil.Universidade Federal do Rio de Janeiro. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Real Time PCR Platform RPT09A. Rio de Janeiro, RJ, Brasil.The emergence of the COVID-19 pandemic resulted in an unprecedented need for RTqPCR- based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the highdemand for molecular diagnostics testing. The timely availability of the complete SARSCoV- 2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RTqPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-82991https://www.arca.fiocruz.br/bitstream/icict/50731/1/license.txt5a560609d32a3863062d77ff32785d58MD51ORIGINALVanessaCosta_VanessaCarnerio_etal_IOC_2021.pdfVanessaCosta_VanessaCarnerio_etal_IOC_2021.pdfapplication/pdf1676111https://www.arca.fiocruz.br/bitstream/icict/50731/2/VanessaCosta_VanessaCarnerio_etal_IOC_2021.pdf1b7f1435b84df1d38aa43aefb148e96bMD52icict/507312022-07-18 16:00:44.104oai:www.arca.fiocruz.br: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ório InstitucionalPUBhttps://www.arca.fiocruz.br/oai/requestrepositorio.arca@fiocruz.bropendoar:21352022-07-18T19:00:44Repositório Institucional da FIOCRUZ (ARCA) - Fundação Oswaldo Cruz (FIOCRUZ)false
dc.title.pt_BR.fl_str_mv Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting
title Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting
spellingShingle Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting
Ferreira, Beatriz Iandra da Silva
Validação
Ensaio molecular
Diagnóstico de COVID-19
Tempor real
PCR
Fusão de alta resolução
Validation
Molecular assay
Diagnostic of COVID-19
Real time PCR
High resolution melting
title_short Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting
title_full Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting
title_fullStr Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting
title_full_unstemmed Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting
title_sort Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting
author Ferreira, Beatriz Iandra da Silva
author_facet Ferreira, Beatriz Iandra da Silva
Gomes, Natália Lins da Silva
Coelho, Wagner Luis da Costa Nunes Pimentel
Costa, Vanessa Duarte da
Carneiro, Vanessa Cristine de Souza
Kader, Rafael Lopes
Amaro, Marisa Pimentel
Villar, Lívia Melo
Miyajima, Fábio
Alves-Leon, Soniza Vieira
Paula, Vanessa Salete de
Leon, Luciane Almeida Amado
Moreira, Otacilio Cruz
author_role author
author2 Gomes, Natália Lins da Silva
Coelho, Wagner Luis da Costa Nunes Pimentel
Costa, Vanessa Duarte da
Carneiro, Vanessa Cristine de Souza
Kader, Rafael Lopes
Amaro, Marisa Pimentel
Villar, Lívia Melo
Miyajima, Fábio
Alves-Leon, Soniza Vieira
Paula, Vanessa Salete de
Leon, Luciane Almeida Amado
Moreira, Otacilio Cruz
author2_role author
author
author
author
author
author
author
author
author
author
author
author
dc.contributor.author.fl_str_mv Ferreira, Beatriz Iandra da Silva
Gomes, Natália Lins da Silva
Coelho, Wagner Luis da Costa Nunes Pimentel
Costa, Vanessa Duarte da
Carneiro, Vanessa Cristine de Souza
Kader, Rafael Lopes
Amaro, Marisa Pimentel
Villar, Lívia Melo
Miyajima, Fábio
Alves-Leon, Soniza Vieira
Paula, Vanessa Salete de
Leon, Luciane Almeida Amado
Moreira, Otacilio Cruz
dc.subject.other.pt_BR.fl_str_mv Validação
Ensaio molecular
Diagnóstico de COVID-19
Tempor real
PCR
Fusão de alta resolução
topic Validação
Ensaio molecular
Diagnóstico de COVID-19
Tempor real
PCR
Fusão de alta resolução
Validation
Molecular assay
Diagnostic of COVID-19
Real time PCR
High resolution melting
dc.subject.en.pt_BR.fl_str_mv Validation
Molecular assay
Diagnostic of COVID-19
Real time PCR
High resolution melting
description Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Real Time PCR Platform RPT09A. Rio de Janeiro, RJ, Brasil.
publishDate 2021
dc.date.issued.fl_str_mv 2021
dc.date.accessioned.fl_str_mv 2022-01-11T18:21:22Z
dc.date.available.fl_str_mv 2022-01-11T18:21:22Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.citation.fl_str_mv FERREIRA, Beatriz Iandra da Silva et al. Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting. Plos One, v. 16, n. 11, p. 1-17, Nov. 2021.
dc.identifier.uri.fl_str_mv https://www.arca.fiocruz.br/handle/icict/50731
dc.identifier.issn.pt_BR.fl_str_mv 1932-6203
dc.identifier.doi.none.fl_str_mv 10.1371/journal.pone.0260087
identifier_str_mv FERREIRA, Beatriz Iandra da Silva et al. Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting. Plos One, v. 16, n. 11, p. 1-17, Nov. 2021.
1932-6203
10.1371/journal.pone.0260087
url https://www.arca.fiocruz.br/handle/icict/50731
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:Repositório Institucional da FIOCRUZ (ARCA)
instname:Fundação Oswaldo Cruz (FIOCRUZ)
instacron:FIOCRUZ
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institution FIOCRUZ
reponame_str Repositório Institucional da FIOCRUZ (ARCA)
collection Repositório Institucional da FIOCRUZ (ARCA)
bitstream.url.fl_str_mv https://www.arca.fiocruz.br/bitstream/icict/50731/1/license.txt
https://www.arca.fiocruz.br/bitstream/icict/50731/2/VanessaCosta_VanessaCarnerio_etal_IOC_2021.pdf
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