Extração e amplificação de RNA do vírus da dengue em microchips de poliéster-toner (PeT)

• Autor(a) principal: Gimenez, Thiago Dadalto
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFG
• dARK ID: ark:/38995/00130000083mc
• Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tede/6000
• topic: Microchips de poliéster-toner; Dengue; RNA; Polyester-toner microchips; QUIMICA::QUIMICA ANALITICA

Miniaturized analytical systems have been successfully used in many analytical and bioanalytical applications. In the field of clinical diagnoses, microfluidic devices can make analyses become faster, cheaper and more sensitive, and can also enable the performance of point-of-care tests. Diagnoses based in molecular biology techniques can be divided in three main steps (nucleic acid extraction, amplification and detection), and all these steps can be adapted for microscale. Here, we report the development of RNA analytical methods in disposable microdevices that may be further used in a micro total analysis system (μTAS). Analytical methods were developed for dengue virus detection, to enable RNA based molecular diagnoses in low cost devices. RNA extraction was carried out through a dynamic solid phase extraction (dSPE) methodology, by using magnetic silica particles. Extraction process occurs by performing three steps: RNA adsorption to the magnetic silica particles surface, washing of the beads and elution of purified RNA. PeT microchips used for RNA extraction were produced by printing, cutting and lamination of polyester films. Microchannels were 18 mm length and 1.2 mm width, and the volume of channels were 6.0 μL. High quality RNA, amplifiable by the reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loopmediated isothermal amplification (RT-LAMP), was obtained by dSPE. In addition to RNA extraction, the RNA detection by RT-LAMP in PeT devices was also demonstrated in this work. Amplified products were separated in agarose gel or detected by naked eye visualization after addition of fluorescent intercalant substances. The success of RNA extraction and amplification by RT-LAMP demonstrates the potential of PeT microdevices in performing molecular diagnosis for detection of pathogens whose genome is consisted by RNA.