Detalhes bibliográficos
| Ano de defesa: |
2022 |
| Autor(a) principal: |
Oliveira, Kézia Gomes de
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| Orientador(a): |
Duarte, Gabriela Rodrigues Mendes
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| Banca de defesa: |
Duarte, Gabriela Rodrigues Mendes,
Chaves, Andrea Rodrigues,
Coltro, Wendell Karlos Tomazelli,
Bailão, Alexandre Melo |
| Tipo de documento: |
Tese
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| 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)
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| Departamento: |
Instituto de Química - IQ (RG)
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| País: |
Brasil
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| Palavras-chave em Português: |
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| Palavras-chave em Inglês: |
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| Área do conhecimento CNPq: |
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| Link de acesso: |
http://repositorio.bc.ufg.br/tede/handle/tede/12256
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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. |
