Acute and chronic sleeping sickness are fatal neglected tropical diseases caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense respectively (members of the sub-genus Trypanozoon). Accurate diagnostics are needed to guide treatment since the symptoms of disease are non-specific and the drugs that are used for treatment are too toxic to be administered to unconfirmed cases. Tests need to be simple enough to confirm clinical diagnosis of sleeping sickness in poorly-resourced, peripheral health centres and for use as epidemiological tools to detect T. b. rhodesiense in the zoonotic reservoirs of infection. This study focuses upon LAMP (loop-mediated isothermal amplification) as a novel diagnostic for sleeping sickness that may serve to bridge the gap between the need for sensitive, specific molecular diagnostics on the one hand and ‘field-friendly’ diagnostics on the other. Here, two previously published LAMP assays for Trypanozoons were compared to classic PCR based methods for the diagnosis of Trypanozoon infection status in 428 cattle blood samples. The results did not support the use of LAMP as an improved system for surveillance of T. b. rhodesiense in the zoonotic cattle reservoir. T. b. rhodesiense and T. b. gambiense subspecies specific LAMP assays were evaluated against traditional reference subspecies specific PCR tests, using DNA purified from 86 cryopreserved trypanosome isolates. Novel LAMP assays for these subspecies were also designed and evaluated. Both the published and novel assays for T. b. rhodesiense (targeting different regions of the SRA gene) were sensitive, specific and reliable when applied to purified DNAs, but were less consistent on field samples. The novel T. b. gambiense LAMP (targeting TgsGP) was sensitive and specific but this was not the case for the published LAMP assay (targeting the 5.8S rRNA gene). However reliability may be less than optimal for LAMP TgsGP. Finally, simple endpoint readout methods for LAMP were evaluated. The colour change reagent hydroxynaphthol blue was identified as the best currently available method taking cost, ease of use and reliability into consideration. In 2009 the number of reported sleeping sickness cases fell below 10,000 for the first time in 50 years. Improved LAMP diagnostics could facilitate the diagnosis of sleeping sickness and support the continued fight against this neglected, but deadly disease.

RNA-cleaving DNAzymes (RCDs) are a class of functional nucleic acids that can bind various targets ranging in size from small molecules to large proteins, which results in activation of cleavage activity. The activation of RCDs results in the cleavage of a ribonucleotide site in an otherwise all-DNA substrate, leading to two cleavage fragments. In this work, a previously identified DNAzyme that binds to a protein biomarker endogenous to Helicobacter pylori (J99) crude extracellular matrix was evaluated for coupling to an isothermal amplification method termed rolling circle amplification (RCA) as a way to improve the originally reported detection limit. Three RCD constructs were designed with the goal of generating a cleavage fragment that could act as a primer to initiate RCA. The first method used the original HP DNAzyme, which liberated a short cleavage fragment that could be used as a primer. However, the primer fragment was rapidly digested by the bacterial matrix, preventing RCA. A second method evaluated use of a circularized substrate and separate RCD to generate a primer, however this system was not capable of generating a cleavage fragment. A final method redesigned the original RCD to move the substrate region from the 3’ to the 5’ end of the RCD, causing the longer RCD-containing fragment to be the primer for RCA. In this case, target-triggered cleavage was observed and the resulting primer was sufficiently resistant to digestion to allow its use as a primer for RCA. Preliminary characterization of the rearranged RCD showed that it retained selectivity similar to the original RCD, but that the cleavage rate was slower. In addition, the RCA based reaction, while successful, did not produce improved detection sensitivity relative to unamplified assays. Methods to further improve RCA performance are discussed for future work. / Thesis / Master of Science (MSc)

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Background: Respiratory Syncytial Virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and children worldwide. Early diagnosis of RSV infection is associated with shorter periods of hospitalisation and decreased mortality. Current point of care (PoC) tests for RSV is less sensitive than molecular methods. Reverse transcription loop-mediated isothermal amplification (RT-LAMP), is a novel method of nucleic acid detection which allows for rapid, robust amplification, and visual detection of infectious agents. Aim: The objective of this study was to develop a novel, rapid, and sensitive multiplex RSV RT-LAMP assay for PoC diagnosis of RSV A and B. Methods: Preparation of a quantitative RSV standard for assay optimisation was done using a rapid hypotonic burst recovery method of infective virus during sub-passaging, and a shell vial fluorescent focus assay for titration of culture-derived viral stock. We designed a single set of eight primers targeting the large polymerase gene of both RSV A and B, and developed a novel single-step multiplex RSV RT-LAMP assay, using an in-house reaction mix and the Rotor-Gene Q real-time thermocycler (Qiagen, Hilden, Germany). The metal ion indicator hydroxy naphtol blue (HNB) was added to the multiplex RSV RT-LAMP assay for visual detection of RSV. Results: The final optimised multiplex RSV RT-LAMP assay had an analytical detection sensitivity of <10 focus forming units (FFU) per reaction for both RSV A and B, with a mean time to positivity of 21.85 minutes (95% CI 19.2-24.5 minutes), compared to 90-120 minutes for conventional PCR. Evaluated against the Seeplex RV15 multiplex PCR (Seegene, Seoul, Korea) by testing 44 (22 RSV A/22 RSV B) nasopharyngeal specimens, the multiplex RSV RT-LAMP assay had a sensitivity of 100%, and a specificity of 100% when screened against nine common respiratory viruses. Visual detection of RSV using HNB as colorimetric reagent was equivalent to the analytical sensitivity (10 FFU/reaction) and specificity (100%) of the multiplex RSV RT-LAMP assay. Conclusion: Compared with conventional PCR, our novel single-step multiplex RSV RTLAMP assay had excellent sensitivity, specificity, and when combined with HNB dye could provide accurate visual diagnosis within 1 hour. We envisage that this multiplex RSV RTLAMP assay will be used for rapid and sensitive RSV detection at the PoC. / AFRIKAANSE OPSOMMING: Agtergrond: Respiratoriese Syncytial Virus (RSV) is die hoof oorsaak van erge laer lugweginfeksie in babas en kinders wêreldwyd. Vroeë diagnose van RSV infeksie word geassosieer met korter periodes van hospitalisasie en verlaagde mortaliteit. Huidige punt van sorg (PoC) toetse vir RSV is minder sensitief as molekulêre metodes. Omgekeerde transkripsie lus-gemedieerde isotermiese amplifisering (RT-LAMP), is 'n nuwe metode van nukleïensuur opsporing wat voorsiening maak vir vinnige, doeltreffende amplifisering, en visuele bevestiging van aansteeklike agente. Doel: Die doel van hierdie studie was om 'n nuwe, vinnige en sensitiewe multipleks RSV RTLAMP toets te ontwikkel wat PoC diagnose van RSV A en B in staat stel. Metodes: Voorbereiding van 'n kwantitatiewe RSV standaard vir toets optimisering is gedoen met behulp van 'n hipotoniese sel-lise metode van infektiewe virus tydens sub-kultuur, en 'n “shell-vial” kultuur en fluorosensie fokus toets vir titrasie van kultuur-geproduseerde virus voorraad. Ons het 'n enkele stel van agt inleiers ontwerp wat gebaseer is op die groot polimerase geen van beide RSV A en B, en 'n nuwe enkel-stap multipleks RSV RT-LAMP toets ontwikkel, met gebruik van 'n in-huis reaksie mengsel en die Rotor-Gene Q “real-time” thermocycler (Qiagen, Hilden, Duitsland). Die metaalioon aanwyser hidroksi naphtol blou (HNB) is bygevoeg in die multipleks RSV RT-LAMP toets vir visuele bevestiging van RSV. Resultate: Die finale geoptimiseerde multipleks RSV RT-LAMP toets het 'n analitiese sensitiwiteit van <10 fokus vormende eenhede (FFU) per reaksie vir beide RSV A en B gehad, met 'n gemiddelde tyd tot positiwiteit van 21.85 minute (95% CI 19.2-24.5 minute) , in vergelyking met 90-120 minute vir konvensionele PCR. Geëvalueer teen die Seeplex RV15 multipleks PCR (Seegene, Seoul, Korea) deur 44 (22 RSV A/22 RSV B) nasofaringeale monsters te toets, het die multipleks RSV RT-LAMP toets 'n sensitiwiteit van 100% getoon, en 'n spesifisiteit van 100% wanneer getoets teen nege algemene respiratoriese virusse. Visuele bevestiging van RSV met gebruik van HNB as kolorimetriese reagens was gelykstaande aan die analitiese sensitiwiteit (10 FFU/reaksie) en spesifisiteit (100%) van die multipleks RSV RT-LAMP toets. Gevolgtrekking: In vergelyking met konvensionele PCR, het ons nuwe enkel-stap multipleks RSV RT-LAMP toets uitstekende sensitiwiteit, spesifisiteit, en wanneer dit gekombineer word met HNB kleurstof kon dit akkurate visuele diagnose voorsien binne 1 uur. Ons verwag dat hierdie multipleks RSV RT-LAMP toets gebruik sal word vir vinnige en sensitiewe RSV bevestiging by die PoC.

<p><a>The hemotrophic mycoplasma<i>, Mycoplasma ovis</i>, is found in sheep and goats throughout the world. This pathogenic bacterium is capable of causing an acute, life-threatening infection as well as chronic or subclinical infections in these animals. The purposes of the present studies were to develop <i>M. ovis</i>-specific assays for detection of this hemoplasma, and to better understand infection dynamics within pregnant ewes and lambs. </a>The first study describes the development and validation of a SYBR^® Green quantitative PCR (qPCR) assay, which was subsequently used to determine the prevalence of <i>M. ovis</i> infection within a population of goats and to evaluate risk factors for infection. This highly sensitive and specific assay consistently detected as few as 10 copies of plasmid/reaction. Convenience-based sampling of 362 goats from 61 farms located in Indiana revealed a prevalence of infection of 18% (95% confidence interval (CI), 14% to 22%). Bacterial loads of <i>M. ovis</i> ranged from 1.05 x 10³ to 1.85 x 10⁵copies/mL of blood with a mean of 1.31 x 10⁴copies/mL of blood. The only risk factor associated with hemoplasma infection was the production use of the goat; dairy goats had a 3.3 fold increase compared with the prevalence in goats used for meat. This study not only demonstrates that <i>M. ovis</i> infection is common in goats in Indiana, but shows the variability of bacterial loads that can be found in chronically-infected animals. While sub-clinically infected goats may have a bacteremia, levels are characteristically less than 2.0 x 10⁵copies/mL.</p><p> The second project utilized a combination of cross-sectional and longitudinal studies to estimate the prevalence of <i>M. ovis</i> infection from a cohort of naturally-infected pregnant ewes, assess changes in their bacterial loads, and determine the incidence of <i>M. ovis</i> in lambs pre- and post-weaning. The prevalence of <i>M. ovis</i> infection in ewes was not found to be significantly different during pregnancy, and before and after weaning of the lambs, with prevalence estimates of 45% (95% CI, 23.1 – 68.5), 36% (95% CI, 17.9 – 57.4), and 44%, (95% CI, 24.4 – 65.1), respectively. Bacterial loads of the ewes from the cross-sectional study ranged from 10⁴to 10⁹copies/mL of blood, with the median bacterial load at 10⁵ copies/mL of blood. While higher bacterial loads are typical of an acute infection, none of the ewes in this study had overt clinical signs. The data suggest that <i>M. ovis</i> loads may be higher in pregnant sheep, particularly in ewes half-way through pregnancy. Most of the <i>M. ovis</i> infections in the study lambs were detected post-weaning which suggests that transplacental or transmammary infection of <i>M. ovis</i> are unlikely routes.</p><p> In the third study, a subset of <i>M. ovis</i> genes for use in a multi-locus sequence typing assay (MLST) were evaluated. Next-generation sequencing was performed to generate data from pooled DNA amplicons in order to identify single nucleotide polymorphisms (SNPs) of <i>M. ovis </i>from five genes. Evaluation of the quality and depths of coverage for the reads and SNPs indicated that the pooled DNA amplicons produced reads and SNPs having high quality and sufficient depth. This pooling technique is a cost-effective alternative to whole-genome sequencing. While the MLST has good discriminatory power and may be used to identify genetically distant and divergent clusters of <i>M. ovis</i> from different geographical origins, within a herd the discrimination power is low, which may hamper its usefulness in transmission studies. </p><p> The fourth and final study was the development of a loop-mediated isothermal amplification (LAMP) assay targeting the dnaK gene of <i>M. ovis</i>, with comparison of the assay to conventional PCR (cPCR). The metal ion indicator hydroxynaphthol blue (HNB) was added prior to the reaction, which allowed for visual detection of LAMP-positive samples as indicated by a color change from violet to sky blue. <i>Mycoplasma ovis</i> was consistently detected in 45 minutes with the LAMP assay at a reaction temperature of 64°C, with more infected sheep being detected than by cPCR. Therefore, the LAMP assay is fast and reliable in the detection of <i>M. ovis</i>. The developed LAMP assay may have applications in diagnostics, surveillance and disease management as well as prevalence studies. However, a more robust molecular technique is necessary for <i>M. ovis</i> isolate or stain discrimination to investigate transmission or disease spread in an outbreak.</p><p> </p><p> In conclusion, three new molecular tools for the detection of <i>M. ovis</i> in goats and sheep were developed as results of these studies. We have shown that the qPCR assay is an efficient tool for detection and quantification of <i>M. ovis</i> loads in blood from both of these species. On the other hand, the value of the LAMP assay is for reliable detection of infection (not quantification), especially in resource-limited situations. The five-locus MLST protocol developed herein, a typing assay based on the polymorphism of five gene sequences, is a laborious technique requiring DNA extraction, PCR amplification, purification and sequencing of target loci. The value of this technique is not as a routine diagnostic, but rather it may be used to better understand the genetic diversity of <i>M. ovis</i> and investigate strain variations. Most importantly, the scheme is sufficiently robust to allow direct genotyping of <i>M. ovis</i> in total blood DNA extracts without culture isolation. The MLST approach may prove useful as a tool for future investigations of transmission and disease spread. These studies have also expanded our understanding of the infection dynamics of <i>M. ovis</i> in pregnant sheep and lambs. It is shown herein that despite the high prevalence and sometimes high bacterial loads in pregnant ewes, <i>M. ovis</i> does not appear to be transmitted to the lambs in utero or during the perinatal period. The lambs become infected mostly after weaning; this may suggest a protective effect during the pre-weaning period and/or subsequent exposure/infection from their environment. </p><br>

A simple, but innovative microfluidic Lab-on-a-chip (LOC) device which is broadly applicable in point of care diagnostics of biological pathogens was designed, fabricated and assembled utilising explicit microfluidic techniques. The purpose of this design was to develop a cartridge with the capability to perform multiplex DNA amplification reactions on a single device. To achieve this outcome, conventional laboratory protocols for sample preparation; involving DNA extraction, purification and elution were miniaturized to suit this lab-on-a-chip device of 75mm X 50mm cross-sectional area. The extraction process was carried out in a uniquely designed microchamber embedded with chitosan membrane that binds DNA at pH 5.0 and elutes when a different solution at pH 9.0 flows through. Likewise, purification protocol that occurs in the designed waste reservoir is very significant in biomedical field because it is concerned with waste treatment and cartridge disposability, was performed with a super absorbent powder that converts liquid to a gel like substance. This powder is known as sodium polyacrylate, which is also they treated with anti-bacterial chemicals to prevent environmental contamination. Furthermore, this process also employed the use of a passive valve for a precise fluid handling operation involving flow regulation from extraction to waste reservoir. In order to achieve the intended multiplexing function a multiplexer was created to distribute flow simultaneously through a bifurcated network of channels connected to six similar amplification microchambers. Prior to fabrication, computational fluid dynamics (CFD) simulation was utilized at flowrates less than 10μL/s as the means to test the effectiveness of each design components and also to specifically deduct empirical values that can be analyzed to improve or understand the relationship between the fluid and geometrical constraints of the microfluidic modular elements. The device produced was a hybrid cartridge composed of PDMS and glass which is the most widely used materials microfluidics research due to their low cost and simplicity of fabrication by soft lithography technique. The choice of material also took into account the various physical and chemical properties advantages and disadvantages in their bio-medical applications. Such properties include but not limited to surface energy that determines the wetting fluid characteristics, biocompatibility, optical transparency. Subsequently, after a prototype cartridge was developed fluid flow experimentation using liquid coloured dye was used on the fully fabricated cartridge to test the efficacy of its microfluidic functionalities before expensive DNA amplification reagents were utilised at similar flowrates to the CFD simulations. This gave rise to comparison between similar and dissimilar flow Peculiarities in the microfluidic circuit of both experiments. The final experiment was performed with the aid of a recent molecular technique in DNA amplification known as of RPA kit (recombinase polymerase amplification reaction). It involved performing two main reaction experiments; first, was the positive experiment that bears the sample DNA and the latter, negative that served as the control without DNA. In the end, quantitative analysis of results was done using an agarose gel that showed 143 base pairs, for the positive samples, thus validating the amplification experiment.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18898号 / 医博第4009号 / 新制||医||1009(附属図書館) / 31849 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木原 正博, 教授 中川 一路, 教授 一山 智 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Final publication is available at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.00270/full / 付記する学位プログラム名：グローバル生存学大学院連携プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19619号 / 医博第4126号 / 新制||医||1015(附属図書館) / 32655 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 木原 正博, 教授 松林 公蔵 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This is an exploration of the potential for a citizen science project, with the goal to get the general public involved in microbial soil biodiversity around Uppsala, Sweden. Biodiversity serves an important role in how an ecosystem performs and functions. A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. It would be beneficial to analyse the abundance and spread of some microorganisms in order to gain a better understanding of soil biodiversity. We suggest that one species family to study could be Phytophthora. Phytophthora is a genus of oomycetes that often are pathogenic, causing disease in various trees and other plants. It is unknown exactly how widespread the genus is today, making it extra interesting for the proposed study. For the general public to be able to do this a device needs to be developed that is easy to use and preferably could be used directly in the field. An isothermal amplification method is suitable for identifying the microorganism under these conditions. Many isothermal amplification methods are expensive, perhaps too expensive for a citizen science study, but have great potential for easy field testing. We propose a device utilizing RPA and lateral flow strips. RPA - Recombinase Polymerase Amplification is a method for amplification that might be suitable since it is simple, sensitive, and has a short run time. It is however expensive, which is an issue, but isothermal amplifications are expensive across the board. Lateral flow strips can be used to visualize the results. They utilize antibodies to detect the previously amplified amplicons, and give a positive or negative test answer that would be understandable to even untrained study participants. One of the biggest obstacles identified in this project concerns amplifying DNA from a soil sample, because an extraction step is necessary. The methods we have identified for extraction are not performable in the field, since they require centrifugation. In the proposition for a device a possible work-around for this is proposed, but since it has yet to be tested it is not yet known whether it will work or not.

Tesis por compendio / [EN] The determination of genetic biomarkers is progressively becoming more extended and popular, being commercialized even in kits for personalized medicine. Establishing specific genotype variations for each patient, such as single nucleotide polymorphisms (SNPs), could be a fundamental tool in the field of diagnosis, prognosis and therapy selection. However, the use of DNA testing is not fully implemented in general healthcare, mainly due to technical and economic barriers associated to the current technologies, which are limited only to specialized centers and large hospitals. In this thesis, the main goal was to overcome these obstacles by developing simpler, faster and more affordable point-of-care (POC) genotyping systems. Allele discrimination was achieved by employing isothermal enzymatic reactions, like recombinase polymerase amplification (RPA), ligation of oligonucleotides and loop-mediated isothermal amplification (LAMP). These processes were integrated to colorimetric indicators and immunoenzymatic assays, in a microarray format. Using compact discs and polycarbonate chips as platforms, the detection was achieved through widespread electronics, like disc-reader, flatbed scanner and smartphone. To demonstrate their capacities, the resulting systems were applied for identifying SNPs in human samples, associated to therapies for tobacco smoking cessation, major depression disorder and blood clotting-related diseases. After selecting the proper conditions, all studied strategies discriminated SNPs in samples containing as low as 100 copies of genomic DNA, with an error rate below 15%. Most importantly, the developed methods have reduced assays times varying between 70 and 140 minutes, at a cost similar to a conventional PCR-based analog, but maintaining or raising amplification efficiency and eliminating the need of specialized temperature cyclers and fluorescence scanners. In conclusion, the biosensors based in isothermal reactions and consumer electronics devices greatly improve the competitivity of POC DNA analysis. It was demonstrated that the technologies developed in this thesis could support genotyping assays in low-resource areas, such as primary healthcare centers and emerging countries. Through this democratization of genetic testing and by performing adequate association studies, molecular diagnostics and personalized medicine practices could have their application extended to the clinical routine. / [ES] La determinación de biomarcadores genéticos es cada vez más extensa y popular, estando incluso comercializándose kits para medicina personalizada. Establecer las variaciones específicas en el genotipo de cada paciente, como los polimorfismos de un solo nucleótido (SNP) podría ser una herramienta fundamental en el campo del diagnóstico, pronóstico y selección de la terapia. Sin embargo, el uso de pruebas de ADN no se encuentra completamente implementado en la atención médica general, principalmente debido a las barreras técnicas y económicas asociadas a las tecnologías actuales, limitadas solamente a centros especializados y grandes hospitales. En esta tesis, el objetivo principal fue superar estos obstáculos mediante el desarrollo de sistemas de genotipado point-of-care (POC), más simples, rápidos y asequibles. La discriminación alélica se logró mediante el uso de reacciones enzimáticas isotermas, como la amplificación de la recombinasa polimerasa (RPA), la ligación de oligonucleótidos y la amplificación isotérmica mediada por bucle (LAMP). Estos procesos se integraron a indicadores colorimétricos y ensayos inmunoenzimáticos en formato de micromatriz. Utilizando discos compactos y chips de policarbonato como plataforma de ensayo, se ha logrado la detección mediante dispositivos electrónicos de consumo, como un lector de discos, escáner documental y teléfono móvil. Para demostrar sus capacidades, los sistemas resultantes se aplicaron a la identificación de SNPs en muestras humanas, asociados a terapias antitabaquismo, para depresión y enfermedades relacionadas con la coagulación de la sangre. Tras seleccionar las condiciones adecuadas, todas las estrategias estudiadas discriminaron SNPs en muestras conteniendo tan solo 100 copias de ADN genómico, con una tasa de error inferior al 15%. Más importante, los métodos desarrollados han reducido los tiempos de ensayo a valores entre 70 y 140 minutos, a un coste similar a un análogo convencional basado en la reacción en cadena de la polimerasa (PCR), pero manteniendo o aumentando la eficiencia de amplificación y eliminando la necesidad de termocicladores y escáneres de fluorescencia. En conclusión, los biosensores basados en reacciones isotérmicas y dispositivos de electrónica de consumo mejoran en gran medida la competitividad del análisis POC de ADN. Se ha demostrado que las tecnologías desarrolladas en esta tesis podrían apoyar los ensayos de genotipado en áreas de recursos escasos, como centros de atención primaria y países emergentes. A través de esta democratización de las pruebas genéticas y realización estudios de asociación adecuados, el diagnóstico molecular y las prácticas en medicina personalizada podrían extender su aplicación a la rutina clínica. / [CA] La determinació de biomarcadors genètics és cada vegada més extensa i popular, estant fins i tot comercialitzant-se kits per a medicina personalitzada. Establir les variacions específiques en el genotip de cada pacient, com els polimorfismes d'un sol nucleòtid (SNP) podria ser una eina fonamental en el camp del diagnòstic, pronòstic i selecció de la teràpia. No obstant això, l'ús de proves d'ADN no es troba completament implementat en l'atenció mèdica general, principalment a causa de les barreres tècniques i econòmiques associades a les tecnologies actuals, limitades solament a centres especialitzats i grans hospitals. En aquesta tesi, l'objectiu principal va ser superar aquests obstacles mitjançant el desenvolupament de sistemes de genotipat point-of-care (POC), més simples, ràpids i assequibles. La discriminació al·lèlica es va aconseguir mitjançant l'ús de reaccions enzimàtiques isotermes, com l'amplificació de la recombinasa polimerasa (RPA), la lligació de oligonucleòtids i l'amplificació isotèrmica mediada per bucle (LAMP). Aquests processos es van integrar a indicadors colorimètrics i assajos inmunoenzimàtics en format de micromatriu. Utilitzant discos compactes i xips de policarbonat com a plataforma d'assaig, s'ha conseguit la detecció mitjançant dispositius electrònics de consum, com un lector de discos, escàner documental i telèfon mòbil. Per a demostrar les seues capacitats, els sistemes resultants es van aplicar a la identificació de polimorfismes en mostres humanes, associats a teràpies antitabaquisme, per a depressió i malalties relacionades amb la coagulació de la sang. Després de seleccionar les condicions adequades, totes les estratègies estudiades van ser capaces de discriminar SNPs en mostres contenint tan sols 100 còpies d'ADN genòmic, amb una taxa d'error inferior al 15%. Més important, els mètodes desenvolupats han reduït els temps d'assaig a valors entre 70 i 140 minuts, a un cost similar a un anàleg convencional basat en la reacció en cadena de la polimerasa (PCR), però mantenint o augmentant l'eficiència d'amplificació i eliminant la necessitat de termocicladors i escàners de fluorescència. En conclusió, els biosensors basats en reaccions isotèrmiques i dispositius d'electrònica de consum milloren en gran manera la competitivitat de l'anàlisi POC del ADN. S'ha demostrat que les tecnologies desenvolupades en aquesta tesi podrien donar suport als assajos de genotipat en àrees de recursos escassos, com a centres d'atenció primària i països emergents. A través d'aquesta democratització de les proves genètiques i realització estudis d'associació adequats, el diagnòstic molecular i les pràctiques en medicina personalitzada podrien estendre la seua aplicació a la rutina clínica. / [PT] A determinação de biomarcadores genéticos está tornando-se cada vez mais extensa e popular, sendo comercializada até em kits para medicina personalizada. O estabelecimento de variações específicas de genotipo para cada paciente, tais como os polimorfismo de nucleotídeo único, pode ser uma ferramenta fundamental no campo do diagnóstico, prognóstico e seleção de terapias. No entanto, o uso de testes de DNA ainda não encontra-se totalmente implementado na área de saúde geral, principalmente devido às barreiras técnicas e econômicas associadas às tecnologias atuais, limitadas apenas a centros especializados e grandes hospitais. Nesta tese, o principal objetivo foi superar esses obstáculos desenvolvendo sistemas de genotipagem point-of-care (POC) de DNA, mais simples, rápidos e acessíveis. A discriminação de alelos foi alcançada empregando reações enzimáticas isotérmicas, como amplificação por recombinase polimerase (RPA), ligação de oligonucleotídeos e amplificação isotérmica mediada por loop (LAMP). Tais processos foram integrados a indicadores colorimétricos e ensaios imunoenzimáticos, em formato micromatriz. Usando discos compactos e chips de policarbonato como plataforma de ensaio, os analitos foram detectados através de dispositivos eletrônicos de consumo, como leitor de disco, scanner de mesa e smartphone. Para demonstrar suas capacidades, os sistemas resultantes foram aplicados para identificação de polimorfismos em amostras de DNA humano, associados a terapias antitabagismo, para depressão e doenças relacionadas à coagulação do sangue. Após a seleção das condições adequadas, todas as estratégias estudadas foram capazes de discriminar SNPs em amostras contendo até 100 cópias de DNA genômico, com uma taxa de erro inferior a 15%. Mais importante, os métodos desenvolvidos reduziram o tempo de ensaio a valores entre 70 e 140 minutos, com um custo similar a um método análogo baseado em reação em cadeia da polimerase (PCR), mas mantendo ou aumentando a eficiência da amplificação e eliminando a necessidade de cicladores de temperatura e scanners de fluorescência especializados. Em conclusão, os biosensores baseados em reações enzimáticas isotérmicas e dispositivos eletrônicos de consumo incrementam grandemente a competitividade da análise POC de DNA. Foi demonstrado que as tecnologias desenvolvidas nesta tese poderiam dar suporte a ensaios de genotipagem em lugares com poucos recursos, como centros de atenção primária e países emergentes. Através desta democratização dos testes genéticos e com a realização de estudos de associação adequados, o diagnóstico molecular e as práticas de medicina personalizada poderiam ter sua aplicação extendida à rotina clínica. / The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 Project and GRISOLIA/2014/024 PhD grant) and the Spanish Ministry of Economy and Competitiveness (MINECO CTQ2013-45875-R project) / Yamanaka, ES. (2020). Isothermal-based DNA biosensors for application in pharmacogenetics [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/148366 / Compendio