Rozprawy doktorskie na temat „Real-Time PCR technique”
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Lintell, Nicholas Adrian, i n/a. "DNA Aberrations in Atypical Cancer Cohorts". Griffith University. School of Biomolecular and Biomedical Science, 2006. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20061009.164402.
Pełny tekst źródłaLintell, Nicholas Adrian. "DNA Aberrations in Atypical Cancer Cohorts". Thesis, Griffith University, 2006. http://hdl.handle.net/10072/365589.
Pełny tekst źródłaThesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text
Netshikweta, Rembuluwani. "Optimisation and assessment of real-time PCR techniques for the detection of selected food- and waterborne viruses". Diss., University of Pretoria, 2011. http://hdl.handle.net/2263/24883.
Pełny tekst źródłaDissertation (MSc)--University of Pretoria, 2011.
Medical Virology
unrestricted
Li, Sichu. "Application of Machine Learning Techniques for Real-time Classification of Sensor Array Data". ScholarWorks@UNO, 2009. http://scholarworks.uno.edu/td/913.
Pełny tekst źródłaSylvester, John T. "Development and evaluation of new techniques to quantify ruminal pool size and duodenal flow of protozoal nitrogen". Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1126015772.
Pełny tekst źródłaTitle from first page of PDF file. Document formatted into pages; contains xviii, 131 p.; also includes graphics. Includes bibliographical references (p. 120-131). Available online via OhioLINK's ETD Center
Smith, Shelle Ann. "A comparison of diagnostic techniques for detecting salmonella spp in equine fecal samples using culture methods, gel-based pcr, and real-time pcr assays". Thesis, Texas A&M University, 2003. http://hdl.handle.net/1969.1/5735.
Pełny tekst źródłaSolliec, Laurent. "Real time flow rate modelling in disturbed conditions from velocity profilers". Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAD052.
Pełny tekst źródłaThe installation of flow rate measurement systems is an important factor in regard to the management of sewer and irrigation networks. Most cities and infrastructure succeed in obtaining sufficient flow measurements to satisfy European Regulation rules. Most flow meters comprise real time systems; this means that the information is permanently transferred to a data base for the management and optimization of the particular network. The measurement technology deployed is typically ultrasound based. Within the number of measurement points a high percentage are often deficient and create specific difficulties (>75% of Venturi flumes are inaccurate according to Anglian Water, a UK water and wastewater company). The study presented here focuses on flow meters which calculate discharge using measurement of level, cross sectional area and the correlation of local velocity to generate a mean value. The aim of this thesis is to propose a real time method to enable determination of this “conversion” under realistic configurations which Users find in open channels. The synthesis of measurement points through an understanding of hydraulic conditions (Bonakdari, 2006) provides a method to create flow data allowing local point velocities to be converted into an overall mean value. The approach has limitations and may fail in industrial situations but can be used for very complex configurations. It also requires specialists with knowledge of the technique who are rarely available to Users. What is proposed here is an alternative method to Bonakdari for simpler configurations. The aim is to evaluate the flow rate with acceptable accuracy using these technics and to establish a relationship between local velocities and the mean velocity according to Regulatory requirements (8% are required in UK, 5 to 8% in Germany depending on area). The individual components are here: the measurement techniques; the hydrodynamics represented with the turbulence (secondary currents in open channels); the wall / roughness effects; the Froude number … for fully developed conditions where conditions become stable in space but for disturbed conditions, as well such as heterogeneous structures or transition conditions
Munõz, Vanessa Nathalia Vargas. "Detection and quantification of Colletotrichum abscissum from leaves of budwood increase block and citrus nursery plants by real time PCR". Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/11/11135/tde-22112018-154045/.
Pełny tekst źródłaO Brasil é o maior produtor de citros do mundo e possui uma grande participação no mercado global de citros. No entanto, várias doenças afetam a cultura, sendo uma delas a podridão floral dos citros (PFC). PFC ganhou importância no Estado de São Paulo pelo deslocamento de áreas de citros para regiões com condições climáticas mais favoráveis para a doença. A identificação precisa do agente causal do PFC foi realizada, tendo sido renomeado como Colletotrichum abscissum. A origem do inóculo inicial ainda é um enigma para as epidemias de PFC e as hipóteses do que o inóculo inicial poderia estar presente no material de propagação já foram discutidas, mas nunca foram demonstradas. O objetivo deste trabalho foi detectar e quantificar Colletotrichum abscissum em folhas de borbulheiras e mudas de citros por meio de qPCR. Neste trabalho, foram utilizadas quatro fazendas comerciais de citros do Estado de São Paulo, Brasil, com borbulheiras e viveiros de mudas de citros das variedades laranja Pera e Valência. C. abscissum foi detectado em borbulheiras e em mudas em ambas as variedades (Valência e Pêra) nas quatro fazendas amostradas. Das 122 amostras de folhas de borbulheiras, 89 (73%) foram positivas para C. absicissum. Das 175 amostras de folhas de mudas de citros, 129 (73%) foram detectadas com o patógeno. A maioria das amostras positivas de borbulheiras e mudas de citros continham 10 a 200 e 10 a 400 conídios de C. absicissum, respectivamente. Com os métodos utilizados, não foi possível isolar o fungo do material vegetativo. Esta descoberta sugere um novo tipo de dispersão a longas distâncias de C. abscissum no ciclo de podridão floral dos citros por meio do material de propagação. A confirmação de C. abscissum nas borbulheiras e mudas de citros levaria à atualização da regulamentação para a produção de mudas de citros certificadas e à busca de novas estratégias de controle do patógeno.
Andrade, Thales Passos de. "DEVELOPMENT AND APPLICATION OF NOVEL QUANTITATIVE AND QUALITATIVE MOLECULAR TECHNIQUES FOR DETECTION OF INFECTIOUS MYONECROSIS VIRUS (IMNV) IN PACIFIC WHITE SHRIMP LITOPENAEUS VANNAMEI". Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/195726.
Pełny tekst źródłaCatalá, García Santiago. "Development of DNA massive sequencing techniques and Real-Time PCR for the detection, identification and quantitation of Phytophthora spp. in environmental samples". Doctoral thesis, Universitat Politècnica de València, 2017. http://hdl.handle.net/10251/90644.
Pełny tekst źródłaEn los últimos años, el aumento del comercio mundial de plantas y el movimiento humano ha promovido el riesgo de introducción de plantas invasoras y patógenos exóticos. Las invasiones biológicas operan a nivel mundial y se consideran como la segunda causa de pérdida de biodiversidad después de la alteración y destrucción directas del hábitat. En este contexto, Phytophthora es uno de los patógenos vegetales más importantes y agresivos en la agricultura y la silvicultura. La detección temprana y la identificación de sus vías son de gran importancia para minimizar la amenaza que representan para los ecosistemas naturales. Se han desarrollado y aplicado diferentes métodos moleculares para la detección de patógenos de plantas en muestras ambientales. Estos métodos permiten una detección e identificación de patógenos rápida y precisa incluso cuando la cantidad de inóculo es baja. Por lo tanto, se propone un nuevo método mejorado para su detección en muestras ambientales a partir de la extracción de ADN ambiental (eDNA) de diferentes fuentes (suelo, raíces y agua) y diferentes ecosistemas. El objetivo del primer capítulo fue aplicar HTS (High Throughput Sequencing) para investigar la presencia de Phytophthora en diferentes comunidades de plantas en bosques naturales, plantaciones y ambientes acuáticos en el norte de España. El eDNA se extrajo del suelo y del agua de los ríos y arroyos de los bosques de Fagus sylvatica y Abies alba y de plantaciones de Chamaecyparis lawsoniana y Pseudotsuga menziesii en el norte de España (bosque de Irati y Villanúa). Se diseñó y aplicó un ensayo específico para la detección de Phytophthora mediante la secuenciación masiva de amplicones basado en la región ITS1. Diferentes valores de threshold se analizaron para la separación óptima de especies de Phytophthora en los análisis bioinformáticos. El agrupamiento al 99% fue el mejor criterio para separar la mayor parte de las especies de Phytophthora. Múltiples Unidades Operacionales Taxonómicas Moleculares (MOTU) correspondientes a 36 especies distintas de Phytophthora se amplificaron en las muestras ambientales. La pirosequenciación de amplicones de muestras de suelo reveló una diversidad baja de Phytophthora (13 especies) en comparación con las 35 especies detectadas en muestras de agua. Trece de los MOTU detectados en los ríos y arroyos no mostraron homología con secuencias depositadas en las bases de datos, lo que revela que la pirosequenciación del ADN ambiental es una estrategia útil para evaluar la diversidad de especies Phytophthora en los ecosistemas naturales. Una vez que la técnica fue desarrollada y validada, se propuso otro objetivo enfocado en el decaimiento de la carrasca. La carrasca (Quercus ilex) es la especie arbórea más representativa de la Península Ibérica y el árbol principal de las dehesas. El decaimiento de la carrasca en suelos no calcáreos en el suroeste de España se ha asociado con Phytophthora cinnamomi durante décadas. Sin embargo, otras especies de Phytophthora como P. quercina y P. psychrophila se han asociado con el declive de Quercus en la parte oriental de España donde predominan los suelos calcáreos. Con el objetivo de investigar la implicación de Phytophthora spp. en el declive de la carrasca en el este de España, se seleccionaron dos bosques en diferentes zonas geográficas (Alcoi y Vallivana) como lugares de muestreo. Las muestras de suelo y raíz se analizaron por pirosequenciación de amplicones. Los resultados de la secuenciación masiva mostraron la diversidad de especies de Phytophthora, y reveló que un taxón nunca aislado de Phytophthora, llamado provisional Phytophthora taxon ballota, fue la especie predominante en ambas áreas. Además, se desarrolló un ensayo de PCR a tiempo real, basado en los resultados de la pirosequenciación, para la detección de este taxón de Phytophthora nunca aislado, y también para la detección de P. quercina
En els últims anys, l'augment del comerç mundial de plantes i el moviment humà ha promogut el risc d'introducció de plantes invasores i patògens exòtics. Les invasions biològiques operen a nivell mundial i es consideren de com la segona causa de pèrdua de biodiversitat després de l'alteració i destrucció directa de l'hàbitat. En aquest context, Phytophthora és un dels mes importants patògens vegetals i agressius en l'agricultura i la silvicultura. La detecció primerenca i la identificació de les seves vies resulten de gran importància per a minimitzar l'amenaça que representen per als ecosistemes naturals. S'han desenvolupat i aplicat diferents mètodes moleculars per a la detecció de patògens de plantes en mostres ambientals. Aquests mètodes permeten una detecció i identificació de patògens ràpida i precisa fins i tot quan la quantitat d'inòcul és baixa. Per tant, es proposa un nou mètode millorat per a la seva detecció en mostres ambientals a partir de l'extracció d'ADN ambiental (eDNA) de diferents fonts (sòl, arrels i aigua) i diferents ecosistemes. L'objectiu del primer capítol va ser aplicar HTS (High Throughput Sequencing) per investigar la presència de Phytophthora en diferents comunitats de plantes en boscos naturals, plantacions i ambients aquàtics al nord d'Espanya. L'eDNA es va extraure del sòl i de l'aigua dels rius i rierols dels boscos de Fagus sylvatica i Abies alba i de plantacions de Chamaecyparis lawsoniana i Pseudotsuga menziesii al nord d'Espanya (bosc d'Irati i Villanúa). Es va dissenyar i va aplicar un assaig específic per a la detecció de Phytophthora mitjançant la seqüenciació massiva de amplicons basat en la regió ITS1. Diferents valors de threshold es van analitzar per a la separació òptima d'espècies de Phytophthora en les anàlisis bioinformàtics. L'agrupament al 99% va ser el millor criteri per separar la major part de les espècies de Phytophthora. Múltiples Unitats Operacionals Taxonòmiques Moleculars (MOTU) corresponents a 36 espècies diferents de Phytophthora es van amplificar en les mostres ambientals. La piroseqüenciació d'amplicons de mostres de sòl va revelar una diversitat baixa de Phytophthora (13 espècies) en comparació amb les 35 espècies detectades en mostres d'aigua. Tretze dels MOTU detectats en els rius i rierols no van mostrar homologia amb seqüències dipositades en les bases de dades, el que revela que la pirosequenciació de l'ADN ambiental és una estratègia útil per avaluar la diversitat d'espècies de Phytophthora en els ecosistemes naturals. Una vegada que la tècnica va ser desenvolupada i validada, es va proposar un altre objectiu enfocat en el decaïment de la carrasca. La carrasca (Quercus ilex) és l'espècie arbòria més representativa de la Península Ibèrica i l'arbre principal de les deveses. El decaïment de la carrasca en sòls no calcaris al sud-oest d'Espanya s'ha associat amb Phytophthora cinnamomi durant dècades. No obstant això, altres espècies de Phytophthora com P. quercina i P. psychrophila s'han associat amb el declivi de Quercus a la part oriental d'Espanya on predominen els sòls calcaris. Amb l'objectiu d'investigar la implicació de Phytophthora spp. en el declivi de la carrasca a l'est d'Espanya, es van seleccionar dos boscos en diferents zones geogràfiques (Alcoi i Vallivana) com a llocs de mostreig. Les mostres de sòl i arrel es van analitzar per piroseqüenciació d'amplicons. Els resultats de la seqüenciació massiva van mostrar la diversitat d'espècies de Phytophthora, i va revelar que un taxó mai aïllat de Phytophthora, anomenat de forma provisional Phytophthora taxon ballota, va ser l'espècie predominant en les dues àrees. A més, es va desenvolupar un assaig de PCR a temps real, basat en els resultats de la piroseqüenciació, per a la detecció d'aquest taxó de Phytophthora mai aïllat, i també per a la detecció de P. quercina. Els assajos de qPCR es van aplicar en mo
Catalá García, S. (2017). Development of DNA massive sequencing techniques and Real-Time PCR for the detection, identification and quantitation of Phytophthora spp. in environmental samples [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90644
TESIS
Rodríguez, Lázaro David. "Development of molecular-based techniques for the detection, identification and quantification of food-borne pathogens". Doctoral thesis, Universitat de Girona, 2004. http://hdl.handle.net/10803/7922.
Pełny tekst źródłaenriquecimientos selectivos, aislamiento en medios selectivos y la confirmación posterior usando pruebas basadas en la morfología, bioquímica y serología propias de cada uno de los microorganismos objeto de estudio. Por lo tanto, estos métodos son laboriosos, requieren un largo proceso para obtener resultados definitivos y, además, no siempre pueden realizarse. Para solucionar estos inconvenientes se han desarrollado diversas metodologías alternativas para la detección identificación y cuantificación de microorganismos patógenos de origen alimentario, entre las que destacan los métodos
inmunológicos y moleculares. En esta última categoría, la técnica basada en la reacción en cadena de la polimerasa (PCR) se ha convertido en la técnica diagnóstica más popular en microbiología, y recientemente, la introducción de una mejora de ésta, la PCR a tiempo real, ha producido una segunda revolución en la metodología diagnóstica molecular, como pude observarse por el número creciente de publicaciones científicas y la aparición continua de nuevos kits comerciales. La PCR a tiempo real es una
técnica altamente sensible -detección de hasta una molécula- que permite la cuantificación exacta de secuencias de ADN específicas de microorganismos patógenos de origen alimentario. Además, otras ventajas que favorecen su implantación potencial en laboratorios de análisis de alimentos son su rapidez, sencillez y el formato en tubo cerrado que puede evitar contaminaciones post-PCR y favorece la automatización y un alto rendimiento.
En este trabajo se han desarrollado técnicas moleculares (PCR y NASBA) sensibles y fiables para la detección, identificación y cuantificación de bacterias patogénicas de origen alimentario (Listeria spp., Mycobacterium avium subsp. paratuberculosis y Salmonella spp.). En concreto, se han diseñado y optimizado métodos basados en la técnica de PCR a tiempo real para cada uno de estos agentes: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, y también se ha optimizado y
evaluado en diferentes centros un método previamente desarrollado para Salmonella spp. Además, se ha diseñado y optimizado un método basado en la técnica NASBA para la detección específica de M. avium subsp. paratuberculosis. También se evaluó la aplicación potencial de la técnica NASBA para la detección específica de formas viables de este microorganismo.
Todos los métodos presentaron una especificidad del 100 % con una sensibilidad adecuada para su aplicación potencial a muestras reales de alimentos. Además, se han desarrollado y evaluado procedimientos de preparación de las muestras en productos cárnicos, productos pesqueros, leche y agua. De esta manera se han desarrollado métodos basados en la PCR a tiempo real totalmente específicos y altamente sensibles para la determinación cuantitativa de L. monocytogenes en productos
cárnicos y en salmón y productos derivados como el salmón ahumado y de M. avium subsp. paratuberculosis en muestras de agua y leche. Además este último método ha sido también aplicado para evaluar la presencia de este microorganismo en el intestino de pacientes con la enfermedad de Crohn's, a partir de biopsias obtenidas de colonoscopia de voluntarios afectados.
En conclusión, este estudio presenta ensayos moleculares selectivos y sensibles para la detección de patógenos en alimentos (Listeria spp., Mycobacterium avium subsp. paratuberculosis) y para una rápida e inambigua identificación de Salmonella spp. La exactitud relativa de los ensayos ha sido excelente, si se comparan con los métodos microbiológicos de referencia y pueden serusados para la cuantificación de tanto ADN genómico como de suspensiones celulares. Por otro lado, la combinación con tratamientos de preamplificación ha resultado ser de gran eficiencia para el análisis de las bacterias objeto de estudio. Por tanto, pueden constituir una estrategia útil para la detección rápida y sensible de patógenos en alimentos y deberían ser una herramienta adicional al rango de herramientas diagnósticas disponibles para el estudio de patógenos de origen alimentario.
The presence of pathogens in foods is among the most serious public health concerns, and the diseases produced by them are a major cause of morbidity. Consequently, the application of microbiological control within the quality assessment programs in the food industry is a premise to minimize the risk of infection for the consumer. Classical microbiological methods involve, in general, the use of a non-selective pre-enrichment, selective enrichment, isolation on selective media, and subsequent confirmation using morphological, biochemical and/or serological tests. Thus, they are laborious, time consuming and not always reliable (e.g. in viable but non-culturable VBNC forms). A number of alternative, rapid and sensitive methods for the detection, identification and quantification of foodborne pathogens have been developed to overcome these drawbacks. PCR has become the most popular microbiological diagnostic method, and recently, the introduction of a development of this technique, RTi-PCR, has produced a second revolution in the molecular diagnostic methodology in microbiology. RTi-PCR is highly sensitive and specific. Moreover, it allows accurate quantification of the bacterial target DNA. Main advantages of RTi-PCR for its application in diagnostic laboratories include quickness, simplicity, the closed-tube format that avoids risks of carryover contaminations and the possibility of high throughput and automation.
In this work, specific, sensitive and reliable analytical methods based on molecular techniques (PCR and NASBA) were developed for the detection, identification and quantification of foodborne pathogens (Listeria spp., Mycobacterium avium subsp. paratuberculosis and Salmonella spp.). Real-time PCR based methods were designed and optimised for each one of these target bacteria: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, and also a real-time PCR based
method previously described for Salmonella spp. was optimised and multicenter evaluated. In addition, an NASBA-based method was designed and optimised for the specific detection of M. avium subsp. paratuberculosis. The potential application of the NASBA technique for specific detection of viable M. avium subsp. paratuberculosis cells was also evaluated.
All the amplification-based methods were 100 % specific and the sensitivity achieved proved to be fully suitable for further application in real food samples. Furthermore, specific pre-amplification procedures were developed and evaluated on meat
products, seafood products, milk and water samples. Thus, fully specific and highly sensitive real-time PCR-based methods were developed for quantitative detection of L. monocytogenes on meat and meat products and on salmon and cold smoked salmon products; and for quantitative detection of M. avium subsp. paratuberculosis on water and milk samples. The M. avium subsp. paratuberculosis-specific real-time PCR-based method was also applied to evaluate the presence of this bacterium in the bowel
of Crohn's disease patients using colonic biopsy specimens form affected and unaffected volunteers. In addition, fully specific and highly sensitive real-time NASBA-based methods were developed for detection of M. avium subsp. paratuberculosis on water and milk samples.
In conclusion, this study reports selective and sensitive amplification-based assays for the quantitative detection of foodborne pathogens (Listeria spp., Mycobacterium avium subsp. paratuberculosis and) and for a quick and unambiguously identification of Salmonella spp. The assays had an excellent relative accuracy compared to microbiological reference methods and can be used for quantification of genomic DNA and also cell suspensions. Besides, in combination with sample pre-amplification treatments,
they work with high efficiency for the quantitative analysis of the target bacteria. Thus, they could be a useful strategy for a quick and sensitive detection of foodborne pathogens in food products and which should be a useful addition to the range of diagnostic tools available for the study of these pathogens.
Quaranta, Giacomo. "Efficient simulation tools for real-time monitoring and control using model order reduction and data-driven techniques". Doctoral thesis, Universitat Politècnica de Catalunya, 2019. http://hdl.handle.net/10803/667474.
Pełny tekst źródłaLa simulación numérica, el uso de ordenadores para ejecutar un programa que implementa un modelo matemático de un sistema físico, es una parte importante del mundo tecnológico actual. En muchos campos de la ciencia y la ingeniería es necesario estudiar el comportamiento de sistemas cuyos modelos matemáticos son demasiado complejos para proporcionar soluciones analíticas, haciendo posible la evaluación virtual de las respuestas de los sistemas (gemelos virtuales). Esto reduce drásticamente el número de pruebas experimentales para los diseños precisos del sistema real que el modelo numérico representa. Sin embargo, estos gemelos virtuales, basados en métodos clásicos que hacen uso de una rica representación del sistema (por ejemplo, el método de elementos finitos), rara vez permiten la retroalimentación en tiempo real, incluso cuando se considera la computación en plataformas de alto rendimiento. En estas circunstancias, el rendimiento en tiempo real requerido en algunas aplicaciones se ve comprometido. En efecto, los gemelos virtuales son estáticos, es decir, se utilizan en el diseño de sistemas complejos y sus componentes, pero no se espera que acomoden o asimilen los datos para definir sistemas de aplicación dinámicos basados en datos. Además, se suelen apreciar desviaciones significativas entre la respuesta observada y la predicha por el modelo, debido a inexactitudes en los modelos empleados, en la determinación de los parámetros del modelo o en su evolución temporal. En esta tesis se proponen diferentes métodos para resolver estas limitaciones con el fin de realizar un seguimiento y un control en tiempo real. En la primera parte se utilizan técnicas de Reducción de Modelos para satisfacer las restricciones en tiempo real; estas técnicas calculan una buena aproximación de la solución simplificando el procedimiento de resolución en lugar del modelo. La precisión de la solución no se ve comprometida y se pueden realizar simulaciones efficientes (gemelos digitales). En la segunda parte se emplea la modelización basada en datos para llenar el vacío entre la solución paramétrica, calculada utilizando técnicas de reducción de modelos no intrusivas, y los campos medidos, con el fin de hacer posibles los sistemas de aplicación dinámicos basados en datos (gemelos híbridos).
La simulation numérique, c'est-à-dire l'utilisation des ordinateurs pour exécuter un programme qui met en oeuvre un modèle mathématique d'un système physique, est une partie importante du monde technologique actuel. Elle est nécessaire dans de nombreux domaines scientifiques et techniques pour étudier le comportement de systèmes dont les modèles mathématiques sont trop complexes pour fournir des solutions analytiques et elle rend possible l'évaluation virtuelle des réponses des systèmes (jumeaux virtuels). Cela réduit considérablement le nombre de tests expérimentaux nécessaires à la conception précise du système réel que le modèle numérique représente. Cependant, ces jumeaux virtuels, basés sur des méthodes classiques qui utilisent une représentation fine du système (ex. méthode des éléments finis), permettent rarement une rétroaction en temps réel, même dans un contexte de calcul haute performance, fonctionnant sur des plates-formes puissantes. Dans ces circonstances, les performances en temps réel requises dans certaines applications sont compromises. En effet, les jumeaux virtuels sont statiques, c'est-à-dire qu'ils sont utilisés dans la conception de systèmes complexes et de leurs composants, mais on ne s'attend pas à ce qu'ils prennent en compte ou assimilent des données afin de définir des systèmes d'application dynamiques pilotés par les données. De plus, des écarts significatifs entre la réponse observée et celle prévue par le modèle sont généralement constatés en raison de l'imprécision des modèles employés, de la détermination des paramètres du modèle ou de leur évolution dans le temps. Dans cette thèse, nous proposons di érentes méthodes pour résoudre ces handicaps afin d'effectuer une surveillance et un contrôle en temps réel. Dans la première partie, les techniques de Réduction de Modèles sont utilisées pour tenir compte des contraintes en temps réel ; elles calculent une bonne approximation de la solution en simplifiant la procédure de résolution plutôt que le modèle. La précision de la solution n'est pas compromise et des simulations e caces peuvent être réalisées (jumeaux numériquex). Dans la deuxième partie, la modélisation pilotée par les données est utilisée pour combler l'écart entre la solution paramétrique calculée, en utilisant des techniques de réduction de modèles non intrusives, et les champs mesurés, afin de rendre possibles des systèmes d'application dynamiques basés sur les données (jumeaux hybrides).
Hamilton, Bryan. "DNA Analysis of Surfactant Associated Bacteria in the Sea Surface Microlayer in Application to Satellite Remote Sensing Techniques: Case Studies in the Straits of Florida and the Gulf of Mexico". NSUWorks, 2015. http://nsuworks.nova.edu/occ_stuetd/39.
Pełny tekst źródłaSillence, Kelly. "Cell-free fetal DNA (cffDNA) enrichment for non-invasive prenatal testing (NIPT) : a comparison of molecular techniques". Thesis, University of Plymouth, 2016. http://hdl.handle.net/10026.1/5319.
Pełny tekst źródłaDe, goussencourt Timothée. "Système multimodal de prévisualisation “on set” pour le cinéma". Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAT106/document.
Pełny tekst źródłaPreviz on-set is a preview step that takes place directly during the shootingphase of a film with special effects. The aim of previz on-set is to show to the film director anassembled view of the final plan in realtime. The work presented in this thesis focuses on aspecific step of the previz : the compositing. This step consists in mixing multiple images tocompose a single and coherent one. In our case, it is to mix computer graphics with an imagefrom the main camera. The objective of this thesis is to propose a system for automaticadjustment of the compositing. The method requires the measurement of the geometry ofthe scene filmed. For this reason, a depth sensor is added to the main camera. The data issent to the computer that executes an algorithm to merge data from depth sensor and themain camera. Through a hardware demonstrator, we formalized an integrated solution in avideo game engine. The experiments gives encouraging results for compositing in real time.Improved results were observed with the introduction of a joint segmentation method usingdepth and color information. The main strength of this work lies in the development of ademonstrator that allowed us to obtain effective algorithms in the field of previz on-set
Ruigrok, Hermanus. "Étude en temps réel des effets cellulaires et moléculaires des champs électromagnétiques radiofréquence environnementaux". Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0674/document.
Pełny tekst źródłaThe biological and health effects of radiofrequency (RF) electromagnetic fields (EMF) exposure have been very actively studied in the past two decades, mainly triggered by concerns about potential health effects of wireless communication systems. This physical agent is among the most common, fastest-growing environmental factors, triggering concerns in the population, as even a minor effect of EMF exposure on health could have a major public health impact. While the effects of extremely low frequency electromagnetic fields (ELF EMF) on the excitation of nerve and muscle cells have been well-characterized, the only well-described effects of radiofrequency electromagnetic fields (RF EMF) on biological systems are caused by dielectric-relaxation heating. In contrast, “nonthermal” RF EMF effects refer to other potential biological effects that are not caused by temperature elevation of living tissue or cell culture medium. The investigation of such mechanisms has been hampered by the absence of robust, reliable and repeatable effects occurring as a consequence of low-level exposures, for which temperature elevation is minimal. Moreover, no plausible mechanistic hypotheses have been given concerning thermal or nonthermal effects of low-level RF EMF exposures, making difficult to draw conclusions on the basis of available experimental results. Nonetheless, in 2011, the International Agency for Research on Cancer (IARC) classified RF emitted by cell phones as “possibly carcinogenic to humans” (Class 2B). The characterization of nonthermal biological RF EMF effects is therefore of primary importance for setting safety limits since guidelines and standards have so far been set to protect from the known health risks associated only with the thermal effects of RF EMF exposures. The aim of this basic science thesis work is to characterize the effects of environmental RF EMF signals on living matter at the cellular and molecular level. In this work, we took advantage of modern and innovative methods to observe the behavior of living matter under RF EMF exposure in real time at various specific absorption rates (SAR). In particular, we have studied: (i) Specific RF EMF effects on the ionic channel TRPV1, a major thermoreceptor in our body. TRPV1 activation under RF EMF exposure was studied using the bioluminescence resonance energy transfer (BRET) technique. The implementation of this technique called for the construction and characterization of BRET probes targeting TRP channels as well as the development of a device for the remote measurement of BRET spectra, using an optical fiber. The conclusion of this part of the thesis is that RFs are able to activate the TRPV1 channel by producing a dielectric heating but in the absence of temperature increase there is no RF effect on the basal activation state of TRPV1 and no change of capsaicin maximal efficacy to activate TRPV1. (ii) The analysis of the global behavior of cells in culture under RF exposure was carried out using a modified xCELLigence system where the array of electrodes of the measuring plates were also used to expose the cells to RF EMF. Using this device, we were able to perform SH-SY5Y cell exposures with a SAR of 24 W/kg without causing heating in the culture medium or in the cell culture. No effect of RF EMF on the behavior of the neuroblastoma SH-SY5Y line could however be demonstrated, either in the absence or in the presence of a co-stimulation by a chemical agent. The conclusion of this study is that under conditions where the temperature remains stable, we have not been able to demonstrate any changes in the functioning of living cells, ether at the molecular level or at the cellular level. The tools developed in this thesis work offer important prospects both in the field of drug screening using spectral BRET, and pave the ways for future studies in bioelectromagnetics
Gualberto, Felipe Augusto Souza. "Valor disgnóstico da nested PCR em tempo real em pacientes com meningite tuberculosa". Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5134/tde-26082014-093325/.
Pełny tekst źródłaBackground: Tuberculous meningitis (TBM) is the most serious and lethal presentation of tuberculosis. Timely diagnosis and appropriated treatment are the main factors associated with good outcome. Methods used in the daily medical practice - clinical, radiological and cerebrospinal fluid (CSF) findings - have low accuracy. Search for Mycobacterium tuberculosis DNA in the CSF by polymerase chain reaction (PCR) using the nested methodology is promising, especially when combined with the practical approach of the real time DNA amplification. Objective: To evaluate the diagnostic value of a nested real-time PCR (nRT-PCR) in the investigation of patients with TBM. Methods: A two-phase observational study was carried out: prospective and retrospective. In the prospective phase, patients with suspected TBM hospitalized at \"Instituto de Infectologia Emílio Ribas\" (IIER) were included. Clinical, laboratory and radiological data were collected, as well as CSF samples of all patients. According to international standard criteria, patients were categorized as \"TBM Definite\", \"TBM Probable\", \"TBM Possible\" and \"Not TBM\". The nRT-PCR, using the mpt64 gene, was performed on all CSF sample in the Laboratory of Bacterial Meningitis, Adolfo Lutz Institute. Sensitivity, specificity and confidence intervals (95% CI) of the nRT-PCR were calculated based on the gold standard (culture positive for M. tuberculosis or AFB isolation on the central nervous system) and on patients with other established diagnoses (\"Not TBM\"). The proportion of patients with a positive nRT-PCR in each clinical category was also calculated. In the retrospective phase, medical chart review was performed in those patients who had the nRT-PCR requested in IIER and in the \"Centro de Referência e Treinamento em DST/AIDS\". The same diagnostic categorization and calculations of sensitivity and specificity were adopted. Results: 102 patients were included in the prospective phase, 92 of them HIV-infected. Nine of them had the gold standard positive and were classified as \"TBM Definite\" and 81 of them had other diagnoses established (\"Not TBM\"). The sensitivity and specificity of the nRT-PCR were 100% (95%CI: 70-100 and 95-100, respectively). The nRT-PCR positivity in category \"TBM Probable\" was 50% (4/8 patients) and 25% in \"TBM Possible\" (1/4). In retrospective phase, the nRT-PCR had a sensitivity of 83% (5/6) and specificity of 100% (0/45), among the 56 included patients (48 of them HIV infected). Positivity in \"TBM Probable\" category was 60% (3/5) and no patients were classified as \"TBM Possible\". Conclusion: The nRT-PCR showed good sensitivity and excellent specificity, showing its diagnostic value in the timely identification of TBM
Mehiaoui, Asma. "Techniques d'analyse et d'optimisation pour la synthèse architecturale de systèmes temps réel embarqués distribués : problèmes de placement, de partitionnement et d'ordonnancement". Thesis, Brest, 2014. http://www.theses.fr/2014BRES0011/document.
Pełny tekst źródłaModern development methodologies from the industry and the academia exploit more and more the ”model” concept to address the complexity of critical real-time systems. These methodologies define a key stage in which the functional model, designed as a network of function blocks communicating through exchanged data signals, is deployed onto a hardware execution platform model and implemented in a software model consisting of a set of tasks and messages. This stage so-called deployment stage allows establishment of an operational architecture of the system, thus it requires evaluation and validation of the temporal properties of the system. In the context of event-driven real-time systems, the verification of temporal properties is performed using the schedulability analysis based on the response time analysis. Each deployment choice has an essential impact on the validity and the quality of the system. However, the existing methodologies do not provide supportto guide the designer of applications in the exploration of the operational architectures space. The objective of this thesis is to develop techniques for analysis and automatic synthesis of a valid operational architecture optimized with respect to the system performances. Our proposition is dedicated to the exploration of architectures space considering at the same time the four degrees of freedom determined during the deployment phase, (i) the placement of functional elements on the computing and communication resources of the execution platform, (ii) the partitioning of function elements into real time tasks and data signals into messages, (iii) the priority assignment to system tasks and messages and (iv) the assignment of shared data protection mechanism for periodic real-time systems. We are mainly interested in meeting temporal constraints and memory capacity of the target platform. In addition, we are focusing on the optimization of end-to-end latency and memory consumption. The design space exploration approaches presented in this thesis are based on the MILP (Mixed Integer Linear programming) optimization technique and concern at the same time time-driven and data-driven applications. Unlike many earlier approaches providing a partial solution to the deployment problem, our methods consider the whole deployment problem. The proposed approaches in this thesis are evaluated using both synthetic and industrial applications
Ninove, Laetitia. "Approche optimisée du diagnostic moléculaire des infections virales : application à la pandémie de grippe A/H1N1". Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX20652/document.
Pełny tekst źródłaMolecular biology techniques have taken an important role in the direct diagnosis of viral pathogens over the last 20 years. Our work focused on establishing and developing a platform for molecular diagnosis in the laboratory of Virology (Timone Hospital) to meet the demands and constraints of diagnosis in hospitals. The organization of this platform required several steps: prevention of contamination risks, aliquoting and storage of reagents, automation techniques of nucleic acid extraction, development of synthetic positive controls and internal controls and optimization of PCR protocols. This optimized approach of the molecular diagnosis of viral infections has particularly been applied to the detection of pandemic influenza A/H1N1v in hospital laboratories for routine and emergency "Point Of Care." The implementation of this platform has significantly improved molecular diagnosis in our laboratory. It currently allows us to detect a large number of pathogens (> 80) and perform tests in a high-throughput (≈ 40,000 tests per year). In total, this platform is at the heart of the laboratory capacity to react quickly to emerging events by rapidly implementing standardized procedures. These techniques have been transferred to many other partners’ laboratories nationally and internationally. We are now considering its use in a syndromic approach including the development of the diagnosis of respiratory viruses
Li, Mengyao. "Approche méthodologique innovante pour le suivi en ligne de procédés de production d’anticorps par cellules animales : apport des techniques spectroscopiques in situ à la stratégie PAT". Thesis, Université de Lorraine, 2018. http://www.theses.fr/2018LORR0151/document.
Pełny tekst źródłaBioprocesses of mammalian cell culture have become essential for the production of therapeutic recombinant proteins, such as monoclonal antibodies (mAb). However, the physiological state of the cells and the quality of the mAb produced, in particular their glycosylation, may vary during the process, and may lead to the alteration of the safety and efficacy of the final product. Consequently, the Process Analytical Technology (PAT) initiative has encouraged the development of online monitoring techniques, with the aim to better control the process and ensure the quality of the final product. In this context, this thesis proposes innovative approaches for online monitoring of CHO (Chinese Hamster Ovary) cells bioreactor cultures, by using three types of in situ spectroscopic measurements (dielectric, Raman, near infrared (NIR)). The first chapter presents a novel approach to predict in real-time one of the major cell physiological state parameters, the specific growth rate (µ). Based on online permittivity measured by in situ dielectric spectroscopy, the cell concentration was estimated and µ was calculated in real-time, making possible to detect the critical moment when µ begins to decrease significantly. Compared to an offline approach, this online approach allowed to maintain the cells in a stable physiological state, ensuring the glycosylation of the mAb produced in feed-harvest cultures. The second chapter shows the use of in situ NIR and Raman spectroscopies combined with chemometric methods. For the first time, the performances of these two spectroscopies were compared in parallel in the same cultures. Online models were developed to predict in real-time the concentration of different parameters (viable cells, glucose, lactate, glutamine, ammonium ions and antibodies). The evaluation of these models by the multivariate Figures of Merit (FOM) revealed some of the advantages of Raman spectroscopy. The combination of the two spectroscopies by various data fusion strategies has also been evaluated. In the third chapter, the interest of Raman spectroscopy for the online monitoring of both the quantity and the glycosylation of the mAb was demonstrated. Models were developed for online prediction of both macroheterogeneity (glycosylation site occupancy) and microheterogeneity (glycan structures) of mAb glycosylation in batch and feed-harvest cultures. The last chapter used models previously developed for NIR and dielectric spectroscopies, to integrate into a “soft sensor” by combining with cell metabolic and mass balance equations. This “soft sensor”, implemented in a fed-batch cell culture for the automatic control of the feed rate, leads to an increased mAb productivity and better mAb glycosylation
Muldur, Sinan. "Développement d’une plateforme immunobiologique microstructurée intégrée à un microscope plasmonique pour le diagnostic de l’inflammation en temps réel". Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1258.
Pełny tekst źródłaState of the art techniques give as a whole the required information needed for the complete cell analysis but require different instruments and different types of platforms. The concept of cells on-a-chip allowing real-time analysis of living cells is, therefore, an important tool for many biomedical research applications such as toxicology and drug discovery. Monitoring in real-time the physical but also chemical response of live cells to specific external stimuli using live-cell imaging can provide a better understanding of the mechanisms and pathways involved in the toxicological reaction. The development of such multianalytical devices for biological analysis relies essentially on the ability to design advanced functional surfaces enabling a controlled interaction and organisation of cells and other nanostructures (e.g antibodies and nanoparticles). Therefore, a large technological effort is related on the development of advanced patterning techniques. In this thesis, we propose two simple and direct micro- and nano-fabrication techniques enabling the creation of cellular and sensing patterns on a non-adhesive and cell repellent plasma-deposited poly (ethyleneoxide) (PEO-like) coating. The first approach consists in immobilising a microarray of ECM molecules (cell-adhesive proteins, e.g fibronectin) on the cell repellent PEO-like surface by physisorption using microspotting or microcontact printing techniques. The second approach enables the creation of Gold nanoparticles (Au NPs) adhesive patterns on the surface using similar spotting techniques. The immobilization of Au NPs on PEO-like coatings does not require any prior chemical modifications and is achieved by a straightforward and irreversible self-assembly technique. These gold nanostructured surfaces have been tested for protein bio-recognition analysis and as a cell culture platform. Ultimately, this platform was integrated to a novel plasmonic microscope which enabled, preliminarily, the label-free monitoring and visualisation of a single cell attachment and detachment in real time, as well as the specific and sensitive detection of test proteins in a cell-free environment
Asmar, Shady. "Diagnostic rapide de la tuberculose par culture". Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM5024.
Pełny tekst źródłaIsolation of Mycobacterium tuberculosis by culture is the gold standard for the diagnosis of tuberculosis. The aim of my thesis work was to simplify and improve the culture diagnosis of tuberculosis. At first we started with a bibliographic study, comparing step by step the different techniques and protocols that have been used for the diagnosis of tuberculosis. This work has allowed us to update our tuberculosis diagnosis protocol, starting with the implementation of a "Tuberculosis-kit" consisting of chlorhexidine containing containers for the recovery and decontamination of non-invasive specimens, followed by culture on an egg-based medium, a micro- colonies detection using an inverted microscope or an automated real-time imaging incubator system and finally an identification using mass spectrometry. We established a new chlorhexidine- based decontamination method that we showed to be more efficient for the recovery and isolation of M. tuberculosis than the standard NALC-NaOH method. Than we developed a new serum-based culture medium, the MOD9 that we showed in a comparative study to be superior to the reference LJ medium for the recovery of M. tuberculosis. In a second study we proved that our chlorhexidine/MOD9 protocol was superior to the standard NALC-NaOH/Bactec 960 MGIT protocol for the isolation of M. tuberculosis. And finally the implementation of a real time imaging system for the detection of M. tuberculosis micro-colonies on MOD9 permits us to dramatically reduce the detection time from 15 days with the standard NALC-NaOH/Bactec 960 MGIT protocol to 3.2 days with our 0.7%-chlorhexidine/MOD9/Advencis-Biosystem protocol with a world record detection time of 25h
Bensmaine, Fayçal. "Modélisation et commande d'un système de stockage d'énergie à base de supercondensateur pour l'hybridation des groupes électrogènes". Thesis, Poitiers, 2014. http://www.theses.fr/2014POIT2341.
Pełny tekst źródłaThe research in this thesis are part of study and control of a new concept of hybrid generator to reduce the power of the diesel engine (downsizing) in order to save fuel and improve the behavior of the synchronous generator during transients. The adopted solution is to place in parallel with the synchronous generator an energy storage system. The latter consists of an inverter with a super capacitor on the DC bus. The aim of the thesis was to scale the entire supercapacitor / static converter and to develop a control law having the best performance with the best compromise between the energy exchanged in the supercapacitor, efficiency, the group speed and voltage amplitude of the generator. A feedback control condition with integration of the deviation using LMI's approach has been established for the synthesis of loop current regulators from the inverter.A second control law was developed to regulate the variable voltage across the supercapacitor. A simulator combining generator and storage system has been developed to test these commands.All validations were made on an experimental test rig specifically developed for this thesis. The tests were conducted with an electric drive motor in the test platform of the LIAS and with a diesel in that of the Leroy Somer Motors company.Finally, experimental tests have highlighted the significant contribution of this hybridization on the diesel speed variationsand on the terminal voltage of the alternator during impact or load shedding
Chang, Hung-An, i 張宏安. "Identification of billfish (Xiphiidae, Istiophoridae) species by a real-time PCR technique". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/54886213523939842122.
Pełny tekst źródła國立臺灣大學
海洋研究所
103
Billfish is an important fishery resource all over the world, and is usually marketed as sashimi, filleted, fish floss and sauce. Most billfish products in Taiwan were only labeled with “billfish” in their ingredients. Therefore, species identification of the billfish products is necessary and will benefit the conservation and regulation of these fishery resourcs. In this study, we developed a real-time PCR approach to identify the six billfish species:Swordfish (Xiphias gladius), Sailfish (Istiophorus platypterus), Black marlin (Istiompax indica), Striped marlin (Kajikia audax), Blue marlin (Makaira nigricans) and Shortbill spearfish (Tetrapturus angustirostris). The whole system contained 14 primers, including 12 species-specific primers and two universal primers designed for discriminating the six billfish from non-billfish species. The 12 species-specific primers showed clear discrimination among the six billfish species, with ΔCt values of the target species ranging between -2.23 ~ 3.93 and the limit of detection is 0.4 ng/μL. In order to minimize the cost and time of whole identification system, we also tested the applicability of the multiplex qPCR by combining several species-specific primers in a single reaction to identify the species. The result showed that the six species can be identified according to the melting curve pattern and Tm values for X. gladius: Tm1 = 75.0 ± 0.0 °C, Tm2 = 83.5 ± 0.0 °C, I. platypterus:Tm1 = 75.2 ± 0.3 °C, Tm2 = 80.5 ± 0.2 °C, I. indica:Tm1 = 77.5 ± 0.0 °C, Tm2 = 80.5 ± 0.0 °C, K. audax:Tm = 80.5 ± 0.0 °C, M. nigricans:Tm1 = 76.0 ± 0.0 °C, Tm2 = 80.5 ± 0.0 °C, T. angustirostris:Tm = 77.0 ± 0.0 °C. An identification protocol for the six billfish species was established in this study. The methods is efficient, sensitive, and cheaper than other conventional methods, which will be helpful for fishery management and protecting the consumer’s rights.
FANG, YEH JU, i 葉如芳. "Microbial Community Dynamics in a Permeable Reactive Barrier using Real-time PCR Technique". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/22789166291578300902.
Pełny tekst źródła大葉大學
環境工程學系碩士班
96
This study was conducted with the application of denaturing gradient gel electrophoresis (DGGE), and real-time quantitative polymerase chain reaction (real-time PCR) molecular biotechnology for monitoring the permeable reactive barrier (PRB) in the relation with BTEX decomposition efficiency and the distribution of microbial community. Various amounts of nitrogen nutrients and BTEX were added to examine the treatment efficiencies. It was shown that the high sodium nitrate amount had improved the BTEX removal, which was an evidence of effects on BTEX treatment. The results of benzene and toluene removal efficiencies revealed that it was completely degraded for both two compounds at the concentrations of 20, 40 and 80 ppm. Increasing the concentrations of these two compounds to 120, 160, 240 and 320 ppm resulted in the decreasing in treatment efficiencies, and the concentrations remained 40, 60, 65, 90 and 100 % for benzene, and 10, 40, 55, 90 % for Toluene, respectively. Furthermore, the microbial variations at various concentrations were consistent via optical density (OD), DGGE analysis and real-time PCR results. Each column test was conducted for 20 days to investigate the effectiveness of oxygen releasing compound (ORC). It was indicated that the highest dissolved oxygen was achieved, which was 5.08 mg/L (equal to 0.25 mg O2/day/g-ORC) at 40 % of CaO2. The results of long-term stability tests of oxygen releasing from PRB system showed that: (1) Oxygen released from ORC was sufficient for the demand of bacteria. (2) In shock-loading of BTEX tests, the removal efficiencies were reduced by 21%, 19%, 17% and 10 % for benzene, toluene, ethylbenzene and xylene, respectively. (3) Removal efficiencies were then recovered in the ascending order of as follow: xylene> ethylbenzene> benzene> toluene. (4) ORC can be used for 40 days. (5) DGGE analysis showed the changing in the microbial community structure before (13 groups) and after shock-loading (reduced to 9 groups), that implied the shock-loading was harmful to bacteria. (6) The results from real-time PCR in the study of catechol 2,3-dioxygenase gene revealed that the quantification of this gene has been declined after shock-loading, but it was latter well again at the 79th day.
Lee, Yi-Shia, i 李宜霞. "Development and Application of the Quantitative Detection of Papaya ringspot potyvirus Based on Real-Time RT-PCR Technique". Thesis, 2006. http://ndltd.ncl.edu.tw/handle/84052872792995182374.
Pełny tekst źródła國立臺灣大學
植物病理與微生物學研究所
94
Papaya ringspot is one of the most destructive diseases of papaya and it is a limiting factor for papaya industry. This disease first occurred in Taiwan in 1975, and it has spreading widely throughout all of papaya-cultivated areas in Taiwan. It is caused by Papaya ringspot virus (PRSV), which belongs to Potyvirus, Potyviridae. According to the different symptoms in papaya hosts, PRSV was divided into three major strains including SM (severe mottling), DF (severe mottling with leaf-deformation) and SMN (severe mottling with necrosis and quick decline) strains. SM was a predominant strain of PRSV in the field of Taiwan several years ago. However, DF and SMN have recently become newly rising dominant strains. Almost full-length of genomic sequences of three PRSV strains were determined in the previous study. Continuous sequencing was conducted in this study. The complete nucleotide sequences (10326 bases) of three strains were determined by application of 5’ RACE and 3’ RACE systems. They encode a polyprotein of 3344 amino acids with a 5’ untranslated region of 85 nucleotides and a 3’ untranslated region of 209 nucleotides. The results of alignment of full genomic nucleotide sequences demonstrated that SM is 97.3% identical to DF, and SMN is 97.0% and 96.8% identical to SM and DF. Most genes in the PRSV genome among the three strains are 95~100% homologous, but the P1 gene and 5’ UTR region are about 95% and 92% homologous among three different strains, respectively. Phylogenic analysis of various international PRSV isolates revealed that SM, DF and SMN are close to the Yung-Kang isolate (another PRSV isolate from Taiwan), but far away from the Thailand and Hawaii isolates. The rapid assay based on reverse transcription-polymerase chain reaction (RT-PCR) with the primer pair PRSV-857 was developed for the detection of PRSV in previous study. Another primer pair PRSV-829 was devised in our study, and it has been proven to achieve better specificity and sensitivity in PRSV detections. In addition, the real-time RT-PCR technology was applied for quantitative monitoring of different PRSV strains in papaya hosts. Both “SYBR Green” and “TaqMan primer/probe” methods were adopted to develop the quantitative detection of PRSV with real-time RT-PCR, and “TaqMan primer/probe” method obtained more satisfactory results. A TaqMan primer/probe combination (named TP-PRSV), selected from the conserved regions of coat protein gene, was designed for the common detections of PRSV. Three primer/probe kits (named TP-DF, TP-SM, and TP-SMN), selected from the variable regions of P1 gene, were also developed for the strain-specific quantitative detection of the DF, SM and SMN strain, respectively. Our devised real-time RT-PCR assays were further applied to monitor the virus multiplicative dynamics in papaya hosts in the inoculation tests with different PRSV strains. When the papaya hosts (cultivar FR) were inoculated with the DF strain, PRSV could be detected 10 days after inoculation. The replication curve (DF) was increasing linearly from the 10th to 16th day, stationary from the 16th to 22th day, and reaching to the peak 24 days after inoculation. When the papayas were inoculated with the SMN strain, PRSV could be detected 10 days after inoculation. The replication curve (SMN) was increasing linearly from the 10th to 18th day, and reaching to the peak 18 days after inoculation. When the papayas were inoculated with both DF and SMN strains, DF could be detected 16 days after inoculation. The replication curve (DF) was increasing linearly from the 16th to 22th day, and reaching to the peak 22 days after inoculation. On the other hand, SMN could be detected 18 days after inoculation. The replication curve (SMN) was increasing linearly from the 18th to 22th day, and reaching to the peak 22 days after inoculation. The development of real-time RT-PCR assays of PRSV is helpful to the ecological studies of papaya ringspot disease.
"Quantitative detection of water-borne bacterial pathogens by filtration, immunomagnetic separation (IMS) and real-time PCR". 2001. http://library.cuhk.edu.hk/record=b5890684.
Pełny tekst źródłaThesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 138-148).
Abstracts in English and Chinese.
Abstract (English) --- p.i
Abstract (Chinese) --- p.iii
Acknowledgements --- p.iv
Abberviations --- p.v
Table of Contents --- p.vii
List of Tables --- p.xiv
List of Figures --- p.xvi
Chapter 1. --- Introduction --- p.1
Chapter 1.1. --- Bacteriological evaluation of water --- p.1
Chapter 1.1.1. --- Indicator organisms for water quality monitoring --- p.2
Chapter 1.1.2. --- Properties defined for indicator organisms --- p.3
Chapter 1.1.3. --- Example of common indicator organisms --- p.3
Chapter 1.1.3.1. --- Total coliform group --- p.3
Chapter 1.1.3.2. --- "Fecal coliform, Escherichia coli" --- p.4
Chapter 1.1.3.3. --- Fecal Streptococcus --- p.5
Chapter 1.1.3.4. --- Klebsiella --- p.5
Chapter 1.2. --- The need for specific detection of waterborne pathogenic organisms --- p.6
Chapter 1.3. --- Common water-borne pathogenic organisms --- p.7
Chapter 1.3.1. --- Bacteria --- p.7
Chapter 1.3.1.1. --- Escherichia coli 0157:H7 --- p.7
Chapter 1.3.1.2. --- Salmonella typhimurium --- p.11
Chapter 1.3.1.3 --- Legionella pneumophila --- p.12
Chapter 1.3.2. --- Protozoa --- p.14
Chapter 1.3.3. --- Viruses --- p.15
Chapter 1.4. --- Conventional approaches for pathogens detection --- p.16
Chapter 1.4.1. --- Examples of conventional detection methods --- p.17
Chapter 1.4.2. --- Problems related to the conventional detection methods --- p.18
Chapter 1.5. --- Novel approaches for pathogens detection --- p.19
Chapter 1.5.1. --- Modifications of media --- p.19
Chapter 1.5.2. --- Antibody-based methods --- p.20
Chapter 1.5.3. --- Nucleic acid-based methods --- p.21
Chapter 1.6. --- Principles of pathogens concentration by filtration and immunomagnetic separation --- p.22
Chapter 1.7. --- Principles of pathogens detection by polymerase chain reaction --- p.24
Chapter 1.8. --- Principles of quantitative assay of water-borne pathogens using real-time PCR --- p.26
Chapter 1.9. --- Aims of this study --- p.28
Chapter 2. --- Detection of water-borne bacteria by polymerase chain reaction --- p.31
Chapter 2.1. --- Introduction --- p.31
Chapter 2.2. --- Materials and Methods --- p.35
Chapter 2.2.1 --- Bacterial strains --- p.35
Chapter 2.2.2. --- Bacterial enumeration --- p.35
Chapter 2.2.3. --- DNA extraction and purification --- p.36
Chapter 2.2.3.1. --- Boiling method --- p.36
Chapter 2.2.3.2. --- Protinesae K extraction method --- p.36
Chapter 2.2.3.3. --- Chelex extraction method --- p.37
Chapter 2.2.4. --- Targeted sequences --- p.38
Chapter 2.2.4.1. --- eaeA gene --- p.38
Chapter 2.2.4.2. --- mdh gene --- p.39
Chapter 2.2.4.3. --- flaR gene --- p.39
Chapter 2.2.5. --- PCR amplification --- p.40
Chapter 2.2.6. --- Gel electrophoresis --- p.41
Chapter 2.3. --- Results --- p.42
Chapter 2.3.1. --- Optimization of the PCR --- p.42
Chapter 2.3.2. --- Sensitivity of PCR detection --- p.42
Chapter 2.3.2.1. --- Boiling method --- p.42
Chapter 2.3.2.2. --- Proteinease K method --- p.43
Chapter 2.3.2.3. --- Chelex method --- p.43
Chapter 2.3.3. --- Specificity of PCR detection --- p.43
Chapter 2.3.3.1. --- primers targeted uidA gene --- p.44
Chapter 2.3.3.2. --- primers targeted mdh gene --- p.44
Chapter 2.3.3.3. --- primers targeted flaR gene --- p.44
Chapter 2.4. --- Discussion --- p.57
Chapter 3. --- Concentration and separation of water-borne bacteria by two-step-filtration and immunomagnetic separation --- p.61
Chapter 3.1. --- Introduction --- p.61
Chapter 3.2. --- Materials and Methods --- p.66
Chapter 3.2.1. --- Bacterial strains --- p.66
Chapter 3.2.2. --- Bacterial enumeration --- p.66
Chapter 3.2.3. --- Filtration --- p.67
Chapter 3.2.4. --- Immunomagnetic separation (IMS) --- p.68
Chapter 3.2.4.1. --- Antibodies and Magnetic beads --- p.68
Chapter 3.2.4.2. --- Binding of antibodies to magnetic beads --- p.68
Chapter 3.2.4.3. --- Immunomagnetic separation of bacteria in seeded samples --- p.70
Chapter 3.2.5. --- Determine the efficiency of filtration and immunomagnetic separation --- p.70
Chapter 3.2.6. --- DNA extraction --- p.71
Chapter 3.2.7. --- Multiplex PCR --- p.71
Chapter 3.2.8. --- PCR amplification --- p.72
Chapter 3.2.9. --- Gel electrophoresis --- p.72
Chapter 3.3. --- Results --- p.73
Chapter 3.3.1. --- Efficiency of filtration and immunomagnetic separation --- p.73
Chapter 3.3.2. --- Detection limit of PCR --- p.73
Chapter 3.3.2.1. --- Filtration and immunomagnetic separation --- p.73
Chapter 3.3.2.2. --- Influence of background flora --- p.73
Chapter 3.3.2.3 --- Shing Mun River and Lam Tsuen River --- p.77
Chapter 3.3.3. --- Multiplex PCR --- p.77
Chapter 3.4. --- Discussion --- p.91
Chapter 4. --- Quantitative assay of water-borne pathogens using real-time PCR --- p.94
Chapter 4.1. --- Introduction --- p.94
Chapter 4.2. --- Materials and Methods --- p.99
Chapter 4.2.1. --- Bacteria strains --- p.99
Chapter 4.2.2. --- Bacterial enumeration --- p.99
Chapter 4.2.3. --- Primers and Probes --- p.100
Chapter 4.2.3.1. --- eaeA gene --- p.101
Chapter 4.2.3.2. --- mdh gene --- p.102
Chapter 4.2.3.3. --- flaR gene --- p.102
Chapter 4.2.4. --- Targeted sequences cloning and sequencing --- p.103
Chapter 4.2.4.1. --- Amplication of targeted sequence by PCR --- p.103
Chapter 4.2.4.2. --- Purification of PCR product --- p.104
Chapter 4.2.4.3. --- Ligation with cloning vector --- p.105
Chapter 4.2.4.4. --- Transformation of E.coli DH5a cells --- p.105
Chapter 4.2.4.5. --- Plasmid DNA isolation --- p.106
Chapter 4.2.4.6. --- DNA quantitation and sequencing --- p.107
Chapter 4.2.5. --- Quantitation determination using real-time PCR --- p.108
Chapter 4.3. --- Results --- p.110
Chapter 4.3.1. --- Determination of targeted sequences --- p.110
Chapter 4.3.2. --- Reading of fluorescence intensity and data analysis --- p.110
Chapter 4.3.3. --- Sensitivity of real-time PCR --- p.114
Chapter 4.3.4. --- Specificity of real-time PCR --- p.121
Chapter 4.3.5. --- Quantitation analysis in seeded samples --- p.121
Chapter 4.4. --- Discussion --- p.131
Chapter 5. --- Conclusion and future perspectives --- p.133
Chapter 6. --- References --- p.138
Liang, Hsin-Yueh, i 梁心玥. "Study of distribution, migration and propagation of Papaya Ringspot Virus in papaya hosts with Real-Time RT-PCR techniques". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/15850407101401998161.
Pełny tekst źródła國立臺灣大學
植物病理與微生物學研究所
97
Papaya ringspot , caused by the Papaya ringspot virus (PRSV), is one of the most important diseases in papaya. PRSV is divided into three major strains distinguished by the symptoms on leaves: severe mottling (SM), deformation (DF), and severe mottling with necrosis (SMN). In this study, three papaya cultivars (lines) were inoculated with PRSV (DF and SMN strain), then the Real-Time RT-PCR assay was used to perform quantitative detection to track the distribution, migration, and propagation of each PRSV in papaya hosts. Three papaya cultivars (lines) were used including Tainung No.2 (TN2), Red Lady (RL), and National Taiwan University Hybrid No.8. (NTU8). After inoculation with the DF strain of PRSV (PRSV/DF), viruses could be detected in roots, stems and leaves of NTU8 within 2 days, which is earliest among the three cultivars. Viruses were found in NTU8 leaves within 4 days post-inoculation, indicating that the viruses moved very fast in NTU8, although they did not replicate well and remained at low quantity (103 copies). PRSV/DF was monitored in roots, stems and leaves of TN2 4, 8 and 10 days after inoculation respectively; the virus quantities in leaves of TN2 reached to a peak (107~8 copies) 24 days later, which is the highest compared to RL and NTU8 . For the RL cultivar, viruses were detected in roots, stems and leaves at 7, 8, and 8 days post-inoculation respectively, and the peak quantity in leaves 24 days after inoculation reached 104 copies. In the inoculation tests with the SMN strain of PRSV (PRSV/SMN), NTU8 exhibited detectable virus levels in roots, stems and leaves 2, 2 and 7 days after inoculation respectively, and the virus quantity remained (103~4 copies) at 24 days later. PRSV/SMN in TN2 was detected in roots and stem after 8 and 4 days respectively, and reached into leaves 10 days after inoculation, where quantity could reach as high as 106 copies after 30 days. The roots, stems and leaves of RL showed detectable virus at 8, 8 and 4 days after inoculation, with quantities at about 104 copies in the leaves 24 days later. In roots usually accumulate more or the same amounts than in leaves, and stems gave rise to slightly lower quantity of viruses than in roots as a results of comparative quantitative detection among roots, stems and leaves no matter what cultivar of papaya was used. It seems that roots are very important for PRSV to propagate. The new bred papaya cultivar tolerant to PRSV, NTU8, constantly showed a good replication of PRSV/DF at 106 copies in roots, but a lower amount of 103 copies in the leaves. TN2, the susceptible cultivar to PRSV, showed more viruses in the leaves approximately 104~5 times than NTU8. This is probably one of the factors resulting in the tolerance of NTU8. When NTU8 was inoculated with PRSV/SMN, it had the virus quantity of 107~8 copies in roots, but only 103~4 copies in leaves; which is similar to the case of PRSV/DF. However, NTU8 seems to have slightly lower tolerance to PRSV/SMN. In this thesis, in addition, the Real-Time RT-PCR method performed its better sensitivity and could detect these viruses at the same time or earlier than the conventional RT-PCR method. Furthermore, this method also provides a precise relative quantification for PRSV and it is helpful to understand how the virus propagates in the host, and will be valuable for future ecological study and control of PRSV.
Huang, Chi-Ming, i 黃啓銘. "Applying quantitative real-time PCR and spore trapping techniques for the development of a rice blast monitoring and forecasting model". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/76684730695635781704.
Pełny tekst źródła國立臺灣大學
植物病理與微生物學研究所
102
Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. In Taiwan, despite the attempt of developing rice blast forecasting model(s) in 1970s, nationwide disease monitoring and notification has long been relying on periodic surveys by trained plant protection personnel. The objective of this study is to first develop an approach which allows the collection and quantification of M. oryzae conidia (airborne inoculum) in the field. A modified blast disease forecasting model, using the amount of conidia along with several weather factors (including temperature, humidity, rainfall, etc.) as parameters, will then be established. We have successfully developed a cyclone-based spore trap and a standard sample processing protocol for extracting DNA from collected airspores. Using quantitative real-time PCR (qPCR) technology and a specific primer pair designed based on a Magnaporthe infection structure specific protein (mif23) gene, the amount of M. oryzae conidia can be easily quantified. While detection limit for the SYBR Green qPCR assay can be as low as 4 copy numbers of M. oryzae gDNA, the limit for reliable and accurate quantification is 10 copy numbers. For the TaqMan assay, the limit for reliable and accurate quantification is 4 copy numbers. Aiming to build a forecasting model, airspore samples, weather data, and disease severity ratings have been periodically collected from ten monitoring stations located at the paddy field and upland field blast nurseries at Chiayi Agricultural Experiment Station, a field site at Chiayi Sikou Farm, and seven field sites chosen by seven District Agricultural Research and Extension Stations in Taiwan (missing data exist for some of the monitoring stations). With our newly-developed spore trap and qPCR technique, M. oryzae spores can be detected before the appearance of leaf blast. It was observed that during the whole season, the amount of spores first increased while the field plants were commonly infected, and it then dropped after the stage of panicle development. In order to improve the handling and storage of airspore samples, we tested the effects of different treatments on the preservation of spore DNA. The optimized way would be: to avoid UV light exposure while sampling, to suspend the sample with CTAB buffer after collection, to store the sample at room temperature or 4℃, and to finish DNA extraction within two weeks. For disease modeling, we developed preliminary rice blast forecasting models for specific rice cultivars (TK9, TN11 and TC192) and multiple cultivars, on the basis of cumulative meteorological data from 1-14 or 7-14 days prior to the prediction day. It was found that the "number of spores" was not considered a significant parameter in most of the models, indicating that weather parameters such as relative humidity and hours of rainfall may be key factors favoring rice blast development. The approaches, sampling ranges and frequencies of the spore trapping and disease ratings may also have some effect on the result. Finally, to make the spore trapping technique applicable for characterization of pathogen physiological races, we developed a high resolution melt (HRM) technique which was proved to be powerful for the detection of the A, D, A+D, and C types of alleles at the pex31 (Avr-pik/kp/km) gene in M. oryzae. The differentiation limit for the HRM analysis is 25 airspores. In the future, with the use of other specific primer pairs, the spore trapping, qPCR, and HRM techniques develop in this study can be widely applied for the monitoring and detection of various airborne diseases. Since the data used for modeling in our study were from the monitoring stations at the Chiayi blast nursery and Sikou Farm, it is important to know that before the forecasting models can be widely applied, more weather data and disease severity data from multiple years, cultivars, and locations are required for model training, validation, and improvement.