Integración de técnicas basadas en ADN para el desarrollo de biosensores aplicados en seguridad alimentaria
[EN] Food security is guaranteed when there is sufficient, safe and nutritious food. This assurance must be satisfied throughout the entire production process, which is known as "safety from farm to fork". This results in a new way of addressing the problem with a global and compre...
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| Tipo de recurso: | tesis doctoral |
| Fecha de publicación: | 2015 |
| País: | España |
| Institución: | Universitat Politècnica de València (UPV) |
| Repositorio: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
| Idioma: | español |
| OAI Identifier: | oai:riunet.upv.es:10251/56464 |
| Acceso en línea: | https://riunet.upv.es/handle/10251/56464 |
| Access Level: | acceso abierto |
| Palabra clave: | Biosensor ADN Amplificación isoterma Microarraying Disco compacto Seguridad alimentaria Screening QUIMICA ANALITICA |
| Sumario: | [EN] Food security is guaranteed when there is sufficient, safe and nutritious food. This assurance must be satisfied throughout the entire production process, which is known as "safety from farm to fork". This results in a new way of addressing the problem with a global and comprehensive approach. To address this challenge, molecular techniques based on the use of nucleic acids are used in the analysis of certain food threatens, such as allergens, microorganisms, genetically modified organisms (GMOs), or food authentication. However, some of the described methods still have limitations, since they are expensive, complicated, and require specialized staff and equipment. Alternatively, biosensor technology provides reliable results in a simpler and faster way and with and added capabilities such as portability and automation, allowing to perform the analysis directly at points-of-control (POC). This thesis has focused on developing a biosensor system, based on compact disc technology, for the detection of nucleic acids in food safety applications and adaptable to POC needs. The carried out investigations have yielded new insights into gene technology, making interesting methodological contributions characterized by miniaturization, integration and automation. The first part of the research deals with the simplification of the amplification step, eluding the thermocycling by using alternatives to the polymerase chain reaction (PCR). To this end, two isothermal amplification techniques have been studied: the recombinase polymerase amplification (RPA) and the multiple displacement amplification (MDA). The detection was performed by hybridization assays with DNA probes immobilized in microarray format on the polycarbonate surface of a DVD. Furthermore, RPA amplification has been combined with detection by an immunoenzymatic assay (ELISA) for the simultaneous detection of multiple analytes. In another approach, amplification and hybridization have been integrated in a single step, further simplifying the analytical process. For that, the isothermal RPA amplification is performed in solid phase on the surface of the disc in different formats: drop, microfluidic chambers or micro-reactors. In the first two formats, the reactions take place at the surface of the DVD and the measurement is performed by recording the intensity of the reflected laser. In the third case the reaction is carried out in micro-wells embedded in the DVD substrate and the measurement is performed by measuring the intensity of the transmitted signal. Finally, a method to real time monitoring DNA synthesis has been developed, integrating the amplification and quantification steps. To this end, the isothermal loop mediated isothermal amplification reaction (LAMP) has been used. The monitoring of the reaction progress is performed by sequentially measuring colorimetric or turbidimetric changes in the reaction mixture. Thus each profile is related to the number of copies of each target gene, allowing their quantitation. The analytical properties (have been established for each methodology and the obtained results have been validated by comparison with reference techniques and by using certified samples. As proof of concept, the different developments have been applied to the detection and determination of the presence of allergens (hazelnut, peanut, soybean, tomato and maize), genetically modified organisms (p35S, tNOS and Bt-11), pathogenic bacteria (Salmonella spp., Cronobacter spp. and Campylobacter spp.), fungi (Fusarium spp.), as well as meat authentication. |
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