New In Vitro Diagnostics for Lactose Intolerance & Legionella Using the Point-Of-Care Platform Doctor Vida

• Autor(a) principal: Conceição, Mariana Martins da
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/146080
• topic: Point-of-Care; Doctor Vida; LAMP; IVD; Intolerância à Lactose; Legionella; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Point-of-care testing (POCT) is a laboratory test that is performed outside a centralized laboratory setting. Loop-mediated isothermal amplification (LAMP) is a popular method for the amplification of nucleic acids in POCT due to its rapidity, high specificity, and robustness. Because it is very efficient under isothermal conditions it can be carried out in devices like the Doctor Vida Pocket (DVPocket). Current molecular methods for the detection of the -13910 C/T polymorphism associated with lactase persistence (LP) rely on invasive samples and require the shipment of samples to centralized laboratories resulting slow turnaround times. The detection of Legionella in water samples, also requires the shipment of samples and the current gold standard is time-consuming and has low sensitivity. To address these limitations a LAMP-based lactose intolerance kit was adapted into the point-of-care (POC) setting by using buccal swabs or capillary blood samples and carrying out the assays in the DVPocket, and validated with both samples from 58 volunteers. Several sample collection and dispensing solutions were assessed to create a user-friendly sample preparation. The kit’s assay maintained its quality in the DVPocket when using buccal swabs and capillary blood, and the overall accuracy was up to 96.5% for buccal swabs and 96.2% for blood. For the Legionella test, two LAMP assays were developed for the detection of Legionella spp. and distinction of L. pneumophila. Assays were performed and optimized in the quantitative polymerase chain reaction (qPCR) instrument, using DNA and inactivated particles, and then performed in the DVPocket. The results showed the detection of Legionella spp. and L. pneumophila in under 1h in the DVPocket. Thus, this study proposes the first POC test for the detection of the -13910 C/T polymorphism in 1h30min and demonstrates the first steps towards the on-site detection of Legionella in water samples, using the POC platform Doctor Vida