Testes moleculares baseados em amplificação isotérmica mediada por Loop para detecção de arbovírus em microdispositivos descartáveis

• Ano de defesa: 2020
• Autor(a) principal: Mendes, Geovana de Melo
• Orientador(a): Duarte, Gabriela Rodrigues Mendes
• Banca de defesa: Duarte, Gabriela Rodrigues Mendes, Sgobbi, Lívia Flório, Assunção, Nilson Antonio de
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Goiás
• Programa de Pós-Graduação: Programa de Pós-graduação em Química (IQ)
• Departamento: Instituto de Química - IQ (RG)
• País: Brasil
• Palavras-chave em Português: RT-LAMP; Dispositivos de PeT; Arbovírus; Diagnóstico molecular; Point-of-care
• Palavras-chave em Inglês: RT-LAMP; PeT microdevice; Arbovirus; Molecular diagnosis; Point-of-care
• Área do conhecimento CNPq: CIENCIAS EXATAS E DA TERRA::QUIMICA
• Link de acesso: http://repositorio.bc.ufg.br/tede/handle/tede/10442

Arboviruses such as dengue virus (DENV) and zika virus (ZIKV) are transmitted to humans through the bite of Aedes mosquitoes. Infections with arboviruses presents similar symptoms and commonly cross reactions. The incidence of these viruses emerges in general in resource limited settings, where sophisticated laboratory infrastructure and trained professionals to make an accurate, reliable and differential diagnosis is not available. Hence, a diagnostic method that are simple, cost effective, reliable and point of care applicable is necessary. In this study we develop assays for the detection of arbovirus in real sample from infected patients by a reverse transcription reaction followed by a loop-mediated isothermal amplification (RT-LAMP) in a polyester toner (PeT) disposable microdevice. The reactions were thermally controlled with a thermoblock and in 10 min it was possible to detect the ZIKV and in 15 min it was possible to detect the DENV. At the end of the incubation time, detection was performed by electrophoresis (off-chip detection) or a DNA fluorescent dye was added for on- chip detection (on-chip detection). Our results demonstrated that it is possible to detect DENV and ZIKV through an RT-LAMP assay directly on samples from infected patients, without a previous RNA extraction step. The success of RT-LAMP was confirmed in reactions initiated with 200 copies of RNA μL-1 for dengue and 0.2 copies of RNA μL-1 for Zika virus. RT-LAMP performed on the PeT microdevice is a simple and inexpensive method that allows for rapid detection of dengue virus with high reliability and great potential for point-of-care applications.