Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis
| Autor(a) principal: | |
|---|---|
| Data de Publicação: | 2022 |
| Outros Autores: | , , , , , , , , , , , , |
| Tipo de documento: | Artigo |
| Idioma: | por |
| Título da fonte: | Repositório Institucional da FIOCRUZ (ARCA) |
| Texto Completo: | https://www.arca.fiocruz.br/handle/icict/52469 |
Resumo: | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil. |
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Manta, Fernanda Saloum de NevesJacomasso, ThiagoRampazzo, Rita de Cássia PontelloMoreira, Suelen Justo MariaZahra, Najua M.Cole, Stewart T.Avanzi, CharlotteLeal-Calvo, ThyagoVasconcellos, Sidra Ezidio GonçalvesSuffys, PhillipRibeiro-Alves, MarceloKrieger, Marco AurelioCosta, Alexandre Dias TavaresMoraes, Milton Ozório2022-04-29T19:30:01Z2022-04-29T19:30:01Z2022MANTA, Fernanda Saloum de Neves et al. Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. PLOS Neglected Tropical Diseases, p. 1–19, 2022.1935-2735https://www.arca.fiocruz.br/handle/icict/5246910.1371/journal. pntd.0009850porPublic Library of ScienceMycobacterium lepraeLeprosyMultiplex Polymerase Chain ReactionReagent Kits, DiagnosticLepraReacción en Cadena de la Polimerasa MultiplexJuego de Reactivos para DiagnósticoLèpreRéaction de polymérisation en chaine multiplexTrousses de réactifs pour diagnosticHanseníaseReação em Cadeia da Polimerase MultiplexKit de Reagentes para DiagnósticoDevelopment and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Global Health Institute. École Polytechnique Fédérale de Lausanne. Lausanne, Switzerland. / Institut Pasteur. Paris, France.Global Health Institute. École Polytechnique Fédérale de Lausanne. Lausanne, Switzerland. / Department of Microbiology, Immunology and Pathology. Mycobacteria Research Laboratories. Colorado State University. Fort Collins, Colorado, United States of America.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação oswaldo Cruz. Instituto Oswaldo Cruz. Laboatório de Biologia Molecular Aplicada a Microbactérias. Rio de Janeiro, RJ, Brasil.Fundação oswaldo Cruz. Instituto Oswaldo Cruz. Laboatório de Biologia Molecular Aplicada a Microbactérias. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Ciências e Tecnologias Aplicadas em Saúde. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Ciências e Tecnologias Aplicadas em Saúde. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and interoperator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.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/52469/1/license.txt783568c2893d2e25a99990b126be1772MD51ORIGINALjournal.pntd.0009850OK.pdfjournal.pntd.0009850OK.pdfapplication/pdf2294689https://www.arca.fiocruz.br/bitstream/icict/52469/2/journal.pntd.0009850OK.pdfe45da6c8b56638e30342328d777b56ccMD52icict/524692022-04-29 16:30:01.21oai:www.arca.fiocruz.br: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ório 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| dc.title.pt_BR.fl_str_mv |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis |
| title |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis |
| spellingShingle |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis Manta, Fernanda Saloum de Neves Mycobacterium leprae Leprosy Multiplex Polymerase Chain Reaction Reagent Kits, Diagnostic Lepra Reacción en Cadena de la Polimerasa Multiplex Juego de Reactivos para Diagnóstico Lèpre Réaction de polymérisation en chaine multiplex Trousses de réactifs pour diagnostic Hanseníase Reação em Cadeia da Polimerase Multiplex Kit de Reagentes para Diagnóstico |
| title_short |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis |
| title_full |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis |
| title_fullStr |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis |
| title_full_unstemmed |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis |
| title_sort |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis |
| author |
Manta, Fernanda Saloum de Neves |
| author_facet |
Manta, Fernanda Saloum de Neves Jacomasso, Thiago Rampazzo, Rita de Cássia Pontello Moreira, Suelen Justo Maria Zahra, Najua M. Cole, Stewart T. Avanzi, Charlotte Leal-Calvo, Thyago Vasconcellos, Sidra Ezidio Gonçalves Suffys, Phillip Ribeiro-Alves, Marcelo Krieger, Marco Aurelio Costa, Alexandre Dias Tavares Moraes, Milton Ozório |
| author_role |
author |
| author2 |
Jacomasso, Thiago Rampazzo, Rita de Cássia Pontello Moreira, Suelen Justo Maria Zahra, Najua M. Cole, Stewart T. Avanzi, Charlotte Leal-Calvo, Thyago Vasconcellos, Sidra Ezidio Gonçalves Suffys, Phillip Ribeiro-Alves, Marcelo Krieger, Marco Aurelio Costa, Alexandre Dias Tavares Moraes, Milton Ozório |
| author2_role |
author author author author author author author author author author author author author |
| dc.contributor.author.fl_str_mv |
Manta, Fernanda Saloum de Neves Jacomasso, Thiago Rampazzo, Rita de Cássia Pontello Moreira, Suelen Justo Maria Zahra, Najua M. Cole, Stewart T. Avanzi, Charlotte Leal-Calvo, Thyago Vasconcellos, Sidra Ezidio Gonçalves Suffys, Phillip Ribeiro-Alves, Marcelo Krieger, Marco Aurelio Costa, Alexandre Dias Tavares Moraes, Milton Ozório |
| dc.subject.other.pt_BR.fl_str_mv |
Mycobacterium leprae |
| topic |
Mycobacterium leprae Leprosy Multiplex Polymerase Chain Reaction Reagent Kits, Diagnostic Lepra Reacción en Cadena de la Polimerasa Multiplex Juego de Reactivos para Diagnóstico Lèpre Réaction de polymérisation en chaine multiplex Trousses de réactifs pour diagnostic Hanseníase Reação em Cadeia da Polimerase Multiplex Kit de Reagentes para Diagnóstico |
| dc.subject.en.pt_BR.fl_str_mv |
Leprosy Multiplex Polymerase Chain Reaction Reagent Kits, Diagnostic |
| dc.subject.es.pt_BR.fl_str_mv |
Lepra Reacción en Cadena de la Polimerasa Multiplex Juego de Reactivos para Diagnóstico |
| dc.subject.fr.pt_BR.fl_str_mv |
Lèpre Réaction de polymérisation en chaine multiplex Trousses de réactifs pour diagnostic |
| dc.subject.decs.pt_BR.fl_str_mv |
Hanseníase Reação em Cadeia da Polimerase Multiplex Kit de Reagentes para Diagnóstico |
| description |
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil. |
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2022 |
| dc.date.accessioned.fl_str_mv |
2022-04-29T19:30:01Z |
| dc.date.available.fl_str_mv |
2022-04-29T19:30:01Z |
| dc.date.issued.fl_str_mv |
2022 |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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MANTA, Fernanda Saloum de Neves et al. Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. PLOS Neglected Tropical Diseases, p. 1–19, 2022. |
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https://www.arca.fiocruz.br/handle/icict/52469 |
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1935-2735 |
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10.1371/journal. pntd.0009850 |
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MANTA, Fernanda Saloum de Neves et al. Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. PLOS Neglected Tropical Diseases, p. 1–19, 2022. 1935-2735 10.1371/journal. pntd.0009850 |
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Public Library of Science |
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Public Library of Science |
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