Análise do efeito do metabolito da análise do efeito do metabolito da microbiota acetato de sódio frente a infecção pelo vírus SARS-COV-2 utilizando metodologia de Lamp

• Autor(a) principal: Reis, Tatiane Madeira
• Data de Publicação: 2022
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da PUC_RS
• Texto Completo: https://tede2.pucrs.br/tede2/handle/tede/10556
• topic: Covid-19; Lamp; PCR em Tempo Real; SARS-CoV-2; Covid-19; Lamp; Real-Time PCR; SARS-CoV-2; CIENCIAS DA SAUDE::MEDICINA; CLINICA MEDICA::PEDIATRIA

The rapid worldwide spread of severe acute respiratory syndrome virus (SARS-CoV-2) infection has been a challenge to science, until there is an effective implementation of a vaccination program, a robust testing strategy, together with prevention measures, will remain the best and most feasible choice for controlling the spread of the disease, so there was a need for new detection methods. The gut microbiota has short-chain fatty acids as its product, one of them is acetate, it protects mice against infection by the VSR strain. However, the role of acetate in SARS-CoV-2 infection is unknown. Many methods for detection of SARS-CoV-2 ribonucleic acid have been developed. The gold standard for RNA detection is reverse transcription quantitative PCR (RT-qPCR), requiring specialized equipment of limited availability in many settings. On the other hand, there are techniques that are increasingly appreciated as suitable for point-of-care testing, among them LAMP (loop-mediated isothermal amplification) which due to their intrinsic simplicity and high sensitivity have an attractive approach. Therefore the study aims to: Test the effects of sodium acetate in controlling SARSCoV-2 infection in vitro and analyze the results using two molecular methodologies. The methodology used in the study includes: cells were pre-treated with 200 µM or 400 µM acetate for 24 h. The cells were infected with 0.1 multiplicity of infection (MOI) of SARS-CoV-2 for another 24 h or 4 days. Cells were harvested for RNA extraction and cDNA synthesis and viral gene expression analysis by real-time PCR. For analysis with another molecular methodology the LAMP methodology was standardized. Initially, the primer sequence was chosen for two genes of SARS-CoV-2. The results were confirmed using an agarose gel, and for quantification, fluorescence measurements were used in a spectrophotometer. The results showed that cells pretreated with acetate at a concentration of 400 µM show less positivity for LAMP-amplified SARS-CoV-2. Conclusion: Our data demonstrate that it is possible to perform a simple LAMP protocol for amplification of SARS-CoV-2 genes.