Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas
Ano de defesa: | 2022 |
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Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | , , , |
Tipo de documento: | Tese |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Federal de Goiás
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Programa de Pós-Graduação: |
Programa de Pós-graduação em Química (IQ)
|
Departamento: |
Instituto de Química - IQ (RG)
|
País: |
Brasil
|
Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://repositorio.bc.ufg.br/tede/handle/tede/12256 |
Resumo: | Infections caused by the new coronavirus (SARS-CoV-2) and arthropod-borne viral infections such as dengue, zika and chikungunya have become a severe public health problem. As all diseases have a significant social impact, the accurate and rapid diagnosis of the infection can be instrumental in treating and adequately controlling the disease. Loop-mediated isothermal amplification (LAMP) emerged in 2000 as an essential alternative to simplify the diagnostics of infectious diseases. An advantage of LAMP is that it allows a straightforward reading of the final result through visual detection. However, this step must be performed with caution to avoid contamination and false-positive results, especially in cases where there is a need to open the tube. In this sense, LAMP performed on microfluidic platforms can minimize false-positive results and have potential for point-of-care applications. Here, we describe a polystyrene (PS-T) centrifugal microfluidic device manually controlled by a hand-spinner for molecular diagnosis of COVID-19, dengue, zika and chikungunya by RT-LAMP, with integrated and automated colorimetric detection. The confirmatory and discriminatory on-chip RT-LAMP test was performed with two types of detection: i) automated addition of SYBR Green fluorescent dye post-amplification (RT-LAMP-SG), and ii) addition of the pH indicator, cresol red, pre-incubation (RT-LAMP-CR). RT-LAMP-SG amplification was performed in a microchamber with a capacity of 5 μL, and the SG was inserted in another chamber with a 3 μL. The reaction was thermally controlled with a thermoblock. At the end of the incubation time (10 min), the detection was performed directly on the device by visual detection after the microdevice spun with a hand-spinner. Our results for the endpoint detection system for LAMP (RT-LAMP-SG) demonstrate that it is possible to detect SARS-CoV-2, DENV-1, ZIKV and CHIKV in the microdevices with a detection limit of approximately 10-3, 660, 30 and 48 RNA copies μL-1 respectively. Clinical samples of patients infected with COVID-19 were tested using our RT-LAMP protocol as well as by conventional RT-qPCR, demonstrating comparable performance to the CDC SARS-CoV-2 RT-qPCR assay. For the RT-LAMP-CR amplification, only the detection of arboviruses was evaluated and presented a detection limit of 1050, 15 and 72.5 copies per μL for detecting DENV-1, ZIKV and CHIKV, respectively. Devices in CD setup were successfully handled performing the three tests simultaneously (dengue, zika and chikungunya) for both detection methods (RT-LAMP-SG and RT-LAMP-CR). The methodologies designated in this study represent simple methods for rapid molecular diagnostics of infectious viral diseases on disposable microdevices, ideal for point-of-care test (POCT) systems. |
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Duarte, Gabriela Rodrigues Mendeshttp://lattes.cnpq.br/9005971441891787Duarte, Gabriela Rodrigues MendesChaves, Andrea RodriguesColtro, Wendell Karlos TomazelliBailão, Alexandre Melohttp://lattes.cnpq.br/7958404744770361Oliveira, Kézia Gomes de2022-08-12T12:50:22Z2022-08-12T12:50:22Z2022-04-14OLIVEIRA, K. G. Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas. 2022. 194 f. Tese (Doutorado em Química) - Universidade Federal de Goiás, Goiânia, 2022.http://repositorio.bc.ufg.br/tede/handle/tede/12256Infections caused by the new coronavirus (SARS-CoV-2) and arthropod-borne viral infections such as dengue, zika and chikungunya have become a severe public health problem. As all diseases have a significant social impact, the accurate and rapid diagnosis of the infection can be instrumental in treating and adequately controlling the disease. Loop-mediated isothermal amplification (LAMP) emerged in 2000 as an essential alternative to simplify the diagnostics of infectious diseases. An advantage of LAMP is that it allows a straightforward reading of the final result through visual detection. However, this step must be performed with caution to avoid contamination and false-positive results, especially in cases where there is a need to open the tube. In this sense, LAMP performed on microfluidic platforms can minimize false-positive results and have potential for point-of-care applications. Here, we describe a polystyrene (PS-T) centrifugal microfluidic device manually controlled by a hand-spinner for molecular diagnosis of COVID-19, dengue, zika and chikungunya by RT-LAMP, with integrated and automated colorimetric detection. The confirmatory and discriminatory on-chip RT-LAMP test was performed with two types of detection: i) automated addition of SYBR Green fluorescent dye post-amplification (RT-LAMP-SG), and ii) addition of the pH indicator, cresol red, pre-incubation (RT-LAMP-CR). RT-LAMP-SG amplification was performed in a microchamber with a capacity of 5 μL, and the SG was inserted in another chamber with a 3 μL. The reaction was thermally controlled with a thermoblock. At the end of the incubation time (10 min), the detection was performed directly on the device by visual detection after the microdevice spun with a hand-spinner. Our results for the endpoint detection system for LAMP (RT-LAMP-SG) demonstrate that it is possible to detect SARS-CoV-2, DENV-1, ZIKV and CHIKV in the microdevices with a detection limit of approximately 10-3, 660, 30 and 48 RNA copies μL-1 respectively. Clinical samples of patients infected with COVID-19 were tested using our RT-LAMP protocol as well as by conventional RT-qPCR, demonstrating comparable performance to the CDC SARS-CoV-2 RT-qPCR assay. For the RT-LAMP-CR amplification, only the detection of arboviruses was evaluated and presented a detection limit of 1050, 15 and 72.5 copies per μL for detecting DENV-1, ZIKV and CHIKV, respectively. Devices in CD setup were successfully handled performing the three tests simultaneously (dengue, zika and chikungunya) for both detection methods (RT-LAMP-SG and RT-LAMP-CR). The methodologies designated in this study represent simple methods for rapid molecular diagnostics of infectious viral diseases on disposable microdevices, ideal for point-of-care test (POCT) systems.Em muitos lugares ao redor do mundo, infecções virais transmitidas por artrópodes como a dengue, zika e chikungunya e infecções virais respiratórias, tal como o SARS-CoV-2 (síndrome respiratória aguda grave de coronavírus 2) tornou-se um grave problema de saúde pública. Assim como para todas as doenças de grande impacto social, o diagnóstico preciso e rápido da infecção, pode ser um grande aliado no tratamento e controle adequado da doença. A amplificação isotérmica mediada por loop (LAMP) emergiu no ano 2000 como uma importante alternativa para simplificar o diagnóstico de doenças infecciosas. Além disso, uma vantagem da LAMP é que ela permite uma leitura fácil do resultado através da detecção visual. No entanto, esta etapa deve ser realizada com cautela para evitar contaminação e resultados falso-positivos, especialmente nos casos em que há necessidade de abrir o tubo ao final da reação. Neste sentido, a LAMP realizada em plataformas microfluidicas pode minimizar resultados falso-positivos, além de ter potencial para aplicações no point-of-care. Neste estudo, descrevemos dispositivos microfluídicos centrífugos de poliestireno (PS-T) controlados por hand-spinner para diagnóstico molecular da COVID-19, dengue, zika e chikungunya por RT-LAMP, com detecção colorimétrica integrada e automatizada. Os testes RT-LAMP on-chip foram realizados com dois tipos de detecção diferentes: i) adição automatizada de SYBR Green pós-amplificação (RT-LAMP-SG), ii) e adição do indicador de pH, vermelho de cresol, pré-incubação (RT-LAMP-VC). A amplificação no teste RT-LAMP-SG foi realizada em uma microcâmara com capacidade de 5 μL e o SG foi adicionado em outra câmara com capacidade para 3 μL, e a reação foi controlada termicamente com um termobloco. Ao final do tempo de incubação (10 min), a detecção foi realizada diretamente no dispositivo por detecção visual após rotação do dispositivo por um hand-spinner. Nossos resultados para os testes com detecção via SYBR Green I demonstram que é possível detectar com sucesso SARS-CoV-2, DENV-1, ZIKV e CHIKV nos microdispositivos e o limite de detecção foi de aproximadamente 10-3 , 660, 30 e 48 cópias do RNA μL-1 respectivamente. Amostras clínicas de COVID-19 foram testadas utilizando o protocolo RT-LAMP-SG, bem como pelo RT-qPCR, demonstrando desempenho comparável ao ensaio CDC para detecção do SARS-CoV-2 RT-qPCR. Para a amplificação RT-LAMP-VC apenas a detecção de arboviroses foi avaliada e apresentou limite de detecção de 1050, 15 e 72,5 cópias μL-1 para detecção do DENV-1, ZIKV e CHIKV, respectivamente. Dispositivos em formato de CD foram utilizados com sucesso para realização dos três testes simultaneamente (dengue, zika e chikungunya) para ambos os métodos de detecção (RT-LAMP-SG e RT-LAMP-VC). As metodologias descritas neste estudo representam métodos simples, utilizadas aqui para diagnósticos moleculares rápidos de doenças virais infecciosas em microdispositivos descartáveis, ideais para sistemas de teste no point-of-care.Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2022-08-11T18:52:41Z No. of bitstreams: 2 Tese - Kézia Gomes de Oliveira - 2022.pdf: 7663703 bytes, checksum: 6415bbdf4985d30b0e44d5185437b0c9 (MD5) license_rdf: 805 bytes, checksum: 4460e5956bc1d1639be9ae6146a50347 (MD5)Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2022-08-12T12:50:22Z (GMT) No. of bitstreams: 2 Tese - Kézia Gomes de Oliveira - 2022.pdf: 7663703 bytes, checksum: 6415bbdf4985d30b0e44d5185437b0c9 (MD5) license_rdf: 805 bytes, checksum: 4460e5956bc1d1639be9ae6146a50347 (MD5)Made available in DSpace on 2022-08-12T12:50:22Z (GMT). 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dc.title.pt_BR.fl_str_mv |
Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas |
dc.title.alternative.eng.fl_str_mv |
Polystyrene-toner microdevice rotationally controlled by a hand-spinner and applications in the molecular diagnosis of diseases infectious |
title |
Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas |
spellingShingle |
Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas Oliveira, Kézia Gomes de RT-LAMP Microdispositivos de PS-T Válvulas hidrofóbicas de toner Hand-spinner PS-T microdevices Hydrophobic toner valves CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA ANALITICA::ELETROANALITICA |
title_short |
Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas |
title_full |
Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas |
title_fullStr |
Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas |
title_full_unstemmed |
Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas |
title_sort |
Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas |
author |
Oliveira, Kézia Gomes de |
author_facet |
Oliveira, Kézia Gomes de |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Duarte, Gabriela Rodrigues Mendes |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/9005971441891787 |
dc.contributor.referee1.fl_str_mv |
Duarte, Gabriela Rodrigues Mendes |
dc.contributor.referee2.fl_str_mv |
Chaves, Andrea Rodrigues |
dc.contributor.referee3.fl_str_mv |
Coltro, Wendell Karlos Tomazelli |
dc.contributor.referee4.fl_str_mv |
Bailão, Alexandre Melo |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/7958404744770361 |
dc.contributor.author.fl_str_mv |
Oliveira, Kézia Gomes de |
contributor_str_mv |
Duarte, Gabriela Rodrigues Mendes Duarte, Gabriela Rodrigues Mendes Chaves, Andrea Rodrigues Coltro, Wendell Karlos Tomazelli Bailão, Alexandre Melo |
dc.subject.por.fl_str_mv |
RT-LAMP Microdispositivos de PS-T Válvulas hidrofóbicas de toner Hand-spinner |
topic |
RT-LAMP Microdispositivos de PS-T Válvulas hidrofóbicas de toner Hand-spinner PS-T microdevices Hydrophobic toner valves CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA ANALITICA::ELETROANALITICA |
dc.subject.eng.fl_str_mv |
PS-T microdevices Hydrophobic toner valves |
dc.subject.cnpq.fl_str_mv |
CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA ANALITICA::ELETROANALITICA |
description |
Infections caused by the new coronavirus (SARS-CoV-2) and arthropod-borne viral infections such as dengue, zika and chikungunya have become a severe public health problem. As all diseases have a significant social impact, the accurate and rapid diagnosis of the infection can be instrumental in treating and adequately controlling the disease. Loop-mediated isothermal amplification (LAMP) emerged in 2000 as an essential alternative to simplify the diagnostics of infectious diseases. An advantage of LAMP is that it allows a straightforward reading of the final result through visual detection. However, this step must be performed with caution to avoid contamination and false-positive results, especially in cases where there is a need to open the tube. In this sense, LAMP performed on microfluidic platforms can minimize false-positive results and have potential for point-of-care applications. Here, we describe a polystyrene (PS-T) centrifugal microfluidic device manually controlled by a hand-spinner for molecular diagnosis of COVID-19, dengue, zika and chikungunya by RT-LAMP, with integrated and automated colorimetric detection. The confirmatory and discriminatory on-chip RT-LAMP test was performed with two types of detection: i) automated addition of SYBR Green fluorescent dye post-amplification (RT-LAMP-SG), and ii) addition of the pH indicator, cresol red, pre-incubation (RT-LAMP-CR). RT-LAMP-SG amplification was performed in a microchamber with a capacity of 5 μL, and the SG was inserted in another chamber with a 3 μL. The reaction was thermally controlled with a thermoblock. At the end of the incubation time (10 min), the detection was performed directly on the device by visual detection after the microdevice spun with a hand-spinner. Our results for the endpoint detection system for LAMP (RT-LAMP-SG) demonstrate that it is possible to detect SARS-CoV-2, DENV-1, ZIKV and CHIKV in the microdevices with a detection limit of approximately 10-3, 660, 30 and 48 RNA copies μL-1 respectively. Clinical samples of patients infected with COVID-19 were tested using our RT-LAMP protocol as well as by conventional RT-qPCR, demonstrating comparable performance to the CDC SARS-CoV-2 RT-qPCR assay. For the RT-LAMP-CR amplification, only the detection of arboviruses was evaluated and presented a detection limit of 1050, 15 and 72.5 copies per μL for detecting DENV-1, ZIKV and CHIKV, respectively. Devices in CD setup were successfully handled performing the three tests simultaneously (dengue, zika and chikungunya) for both detection methods (RT-LAMP-SG and RT-LAMP-CR). The methodologies designated in this study represent simple methods for rapid molecular diagnostics of infectious viral diseases on disposable microdevices, ideal for point-of-care test (POCT) systems. |
publishDate |
2022 |
dc.date.accessioned.fl_str_mv |
2022-08-12T12:50:22Z |
dc.date.available.fl_str_mv |
2022-08-12T12:50:22Z |
dc.date.issued.fl_str_mv |
2022-04-14 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
OLIVEIRA, K. G. Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas. 2022. 194 f. Tese (Doutorado em Química) - Universidade Federal de Goiás, Goiânia, 2022. |
dc.identifier.uri.fl_str_mv |
http://repositorio.bc.ufg.br/tede/handle/tede/12256 |
identifier_str_mv |
OLIVEIRA, K. G. Microdispositivo de poliestireno-toner rotacionalmente controlado por um hand-spinner e aplicações no diagnóstico molecular de doenças infecciosas. 2022. 194 f. Tese (Doutorado em Química) - Universidade Federal de Goiás, Goiânia, 2022. |
url |
http://repositorio.bc.ufg.br/tede/handle/tede/12256 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.program.fl_str_mv |
84 |
dc.relation.confidence.fl_str_mv |
500 500 500 500 500 |
dc.relation.department.fl_str_mv |
29 |
dc.relation.cnpq.fl_str_mv |
1069 |
dc.relation.sponsorship.fl_str_mv |
1 3 |
dc.rights.driver.fl_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
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Universidade Federal de Goiás |
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Programa de Pós-graduação em Química (IQ) |
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UFG |
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Brasil |
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Instituto de Química - IQ (RG) |
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Universidade Federal de Goiás |
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tasesdissertacoes.bc@ufg.br |
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