Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study

Detalhes bibliográficos
Autor(a) principal: Ali, Nasir
Data de Publicação: 2020
Outros Autores: Bello, Graziele Lima, Rossetti, Maria Lúcia Rosa, Krieger, Marco Aurélio, Tavares, Alexandre Dias
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
Texto Completo: https://www.arca.fiocruz.br/handle/icict/45735
Resumo: Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Departamento de Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Departamento de Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil.
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spelling Ali, NasirBello, Graziele LimaRossetti, Maria Lúcia RosaKrieger, Marco AurélioTavares, Alexandre Dias2021-01-19T14:19:58Z2021-01-19T14:19:58Z2020ALI, Nasir et al. Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study. PLoS ONE, v.15, n. 12, p. 1–16, 2020.1932-6203https://www.arca.fiocruz.br/handle/icict/45735doi.org/10.1371/journal.pone.0242408engPublic Library of ScienceqPCRMycobacterium tuberculosis H37RvReal-Time Polymerase Chain ReactionClinical ProtocolsLiquid-Liquid ExtractionMycobacterium tuberculosisReacción en Cadena en Tiempo Real de la PolimerasaProtocolos ClínicosExtracción Líquido-LíquidoADNMycobacterium tuberculosisReacción en Cadena en Tiempo Real de la PolimerasaProtocoles cliniquesExtraction liquide-liquideADNMycobacterium tuberculosisReação em Cadeia da Polimerase em Tempo RealProtocolos ClínicosExtracción Líquido-LíquidoDNADemonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept studyinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Departamento de Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Departamento de Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil.Secretaria Estadual de Saúde. Centro de Desenvolvimento Científico e Tecnológico. Porto Alegre, RS, Brasil. / Universidade Luterana do Brasil. Programa de Pós-Graduação em Biologia Celular e Molecular Aplicado à Saúde. Canoas, RS, Brasil.Secretaria Estadual de Saúde. Centro de Desenvolvimento Científico e Tecnológico. Porto Alegre, RS, Brasil. / Universidade Luterana do Brasil. Programa de Pós-Graduação em Biologia Celular e Molecular Aplicado à Saúde. Canoas, RS, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Departamento de Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucinbased matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95˚C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof–of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-83084https://www.arca.fiocruz.br/bitstream/icict/45735/1/license.txt783568c2893d2e25a99990b126be1772MD51ORIGINALpone.0242408OK.pdfpone.0242408OK.pdfapplication/pdf924516https://www.arca.fiocruz.br/bitstream/icict/45735/2/pone.0242408OK.pdf24d3b2548ffb8ce1f29f559e423f4e3aMD52TEXTpone.0242408OK.pdf.txtpone.0242408OK.pdf.txtExtracted texttext/plain61046https://www.arca.fiocruz.br/bitstream/icict/45735/3/pone.0242408OK.pdf.txtc6f62ab157b2008555c8694659c98d7dMD53icict/457352021-01-20 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dc.title.pt_BR.fl_str_mv Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study
title Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study
spellingShingle Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study
Ali, Nasir
qPCR
Mycobacterium tuberculosis H37Rv
Real-Time Polymerase Chain Reaction
Clinical Protocols
Liquid-Liquid Extraction
Mycobacterium tuberculosis
Reacción en Cadena en Tiempo Real de la Polimerasa
Protocolos Clínicos
Extracción Líquido-Líquido
ADN
Mycobacterium tuberculosis
Reacción en Cadena en Tiempo Real de la Polimerasa
Protocoles cliniques
Extraction liquide-liquide
ADN
Mycobacterium tuberculosis
Reação em Cadeia da Polimerase em Tempo Real
Protocolos Clínicos
Extracción Líquido-Líquido
DNA
title_short Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study
title_full Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study
title_fullStr Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study
title_full_unstemmed Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study
title_sort Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study
author Ali, Nasir
author_facet Ali, Nasir
Bello, Graziele Lima
Rossetti, Maria Lúcia Rosa
Krieger, Marco Aurélio
Tavares, Alexandre Dias
author_role author
author2 Bello, Graziele Lima
Rossetti, Maria Lúcia Rosa
Krieger, Marco Aurélio
Tavares, Alexandre Dias
author2_role author
author
author
author
dc.contributor.author.fl_str_mv Ali, Nasir
Bello, Graziele Lima
Rossetti, Maria Lúcia Rosa
Krieger, Marco Aurélio
Tavares, Alexandre Dias
dc.subject.other.pt_BR.fl_str_mv qPCR
topic qPCR
Mycobacterium tuberculosis H37Rv
Real-Time Polymerase Chain Reaction
Clinical Protocols
Liquid-Liquid Extraction
Mycobacterium tuberculosis
Reacción en Cadena en Tiempo Real de la Polimerasa
Protocolos Clínicos
Extracción Líquido-Líquido
ADN
Mycobacterium tuberculosis
Reacción en Cadena en Tiempo Real de la Polimerasa
Protocoles cliniques
Extraction liquide-liquide
ADN
Mycobacterium tuberculosis
Reação em Cadeia da Polimerase em Tempo Real
Protocolos Clínicos
Extracción Líquido-Líquido
DNA
dc.subject.en.pt_BR.fl_str_mv Mycobacterium tuberculosis H37Rv
Real-Time Polymerase Chain Reaction
Clinical Protocols
Liquid-Liquid Extraction
dc.subject.es.pt_BR.fl_str_mv Mycobacterium tuberculosis
Reacción en Cadena en Tiempo Real de la Polimerasa
Protocolos Clínicos
Extracción Líquido-Líquido
ADN
dc.subject.fr.pt_BR.fl_str_mv Mycobacterium tuberculosis
Reacción en Cadena en Tiempo Real de la Polimerasa
Protocoles cliniques
Extraction liquide-liquide
ADN
dc.subject.decs.pt_BR.fl_str_mv Mycobacterium tuberculosis
Reação em Cadeia da Polimerase em Tempo Real
Protocolos Clínicos
Extracción Líquido-Líquido
DNA
description Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Departamento de Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Departamento de Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil.
publishDate 2020
dc.date.issued.fl_str_mv 2020
dc.date.accessioned.fl_str_mv 2021-01-19T14:19:58Z
dc.date.available.fl_str_mv 2021-01-19T14:19:58Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
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status_str publishedVersion
dc.identifier.citation.fl_str_mv ALI, Nasir et al. Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study. PLoS ONE, v.15, n. 12, p. 1–16, 2020.
dc.identifier.uri.fl_str_mv https://www.arca.fiocruz.br/handle/icict/45735
dc.identifier.issn.pt_BR.fl_str_mv 1932-6203
dc.identifier.doi.none.fl_str_mv doi.org/10.1371/journal.pone.0242408
identifier_str_mv ALI, Nasir et al. Demonstration of a fast and easy sample-toanswer protocol for tuberculosis screening in point-of-care settings: A proof of concept study. PLoS ONE, v.15, n. 12, p. 1–16, 2020.
1932-6203
doi.org/10.1371/journal.pone.0242408
url https://www.arca.fiocruz.br/handle/icict/45735
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
instname_str Fundação Oswaldo Cruz (FIOCRUZ)
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institution FIOCRUZ
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