The amount of information derived from sequencing the humangenome is leading to an exponential increase in the rate atwhich genes and genetic disorders are mapped and characterized.As a consequence, the demand for genetic testing is alsodramatically increasing. Screening and assaying methods, otherthan direct sequencing, are gradually becoming available,although with different robustness, sensitivity and throughput.In the work summarized in this thesis, attention was devotedprimarily to the improvement and the development of newtechniques for some of these methods. Considering the role of capillary electrophoresis inmolecular diagnostics and an associated important phenomenon -electroosmotic flow (EOF) - a robust, reproducible procedurefor surface modification of the inner wall of capillaries wasreported (Paper I) and this method was beneficially employed insubsequent projects. The effort to screen and characterize point mutations in theCACNA1A gene, responsible for the Familial Hemiplegic Migraine(FHM) disease, led to the optimization and validation of a verysensitive technique on slab gel called Double Gradient–Denaturing Gradient Gel Electrophoresis (DG-DGGE) (Paper II). A more reliable and robust method forSingle-Strand Conformation Polymorphism analysis (SSCP) bycapillary electrophoresis, making use of neutral pH buffers,was also developed (Paper III), while the next project resulted in thedevelopment of a high-throughput method for assaying knownpolymorphisms by multiplex solid-phase minisequencing inmulti-capillary format using a detection system based on liquidcore waveguiding (Paper IV). As these and other methods, as well as most applications inmolecular diagnostics and molecular biology, depend on thepolymerase chain reaction (PCR), an effort was made to enhancethe throughput of this technology and to minimize reactionvolumes and costs. For this, the concept of a dynamic reactorwas employed, instead of static systems where the reactionmixture is exposed to temperature cycles in a confined space. Acontinuous flow of small-volume reaction mixtures, separated byan immiscible hydrophobic carrier fluid such as aperfluorocarbon, is transported in a hydrophobic tube throughthree zones, which are kept at constant temperatures optimizedfor denaturation, annealing and elongation (Paper V). If combined with a technique for automatedsample loading and collection (Chapter 7), this method should allow veryhigh-throughput miniaturized DNA amplification. A samplehandling concept using hydrophilic anchors is proposed, whichshould also be useful for other miniaturized reactions andchemical processing. Finally, some possible alternative methods are discussed aswell as future trends. <b>Keywords:</b>Amplification, anchor, array, assay,capillary, DGGE, disease, disorder, DNA, droplet, dynamicreactor, electrophoresis, fluorescence, fluorocarbon, gel,genetic alteration, genomics, genotyping, high-throughput,hydrophobic, liquid lid, miniaturized reaction, minisequencing,molecular diagnostics, mutation, PCR, polymerase chainreaction, screening, segmented flow, single nucleotidepolymorphism, SNP, SSCP, test.

<p>The amount of information derived from sequencing the humangenome is leading to an exponential increase in the rate atwhich genes and genetic disorders are mapped and characterized.As a consequence, the demand for genetic testing is alsodramatically increasing. Screening and assaying methods, otherthan direct sequencing, are gradually becoming available,although with different robustness, sensitivity and throughput.In the work summarized in this thesis, attention was devotedprimarily to the improvement and the development of newtechniques for some of these methods.</p><p>Considering the role of capillary electrophoresis inmolecular diagnostics and an associated important phenomenon -electroosmotic flow (EOF) - a robust, reproducible procedurefor surface modification of the inner wall of capillaries wasreported (<i>Paper I</i>) and this method was beneficially employed insubsequent projects.</p><p>The effort to screen and characterize point mutations in theCACNA1A gene, responsible for the Familial Hemiplegic Migraine(FHM) disease, led to the optimization and validation of a verysensitive technique on slab gel called Double Gradient–Denaturing Gradient Gel Electrophoresis (DG-DGGE) (<i>Paper II</i>). A more reliable and robust method forSingle-Strand Conformation Polymorphism analysis (SSCP) bycapillary electrophoresis, making use of neutral pH buffers,was also developed (<i>Paper III</i>), while the next project resulted in thedevelopment of a high-throughput method for assaying knownpolymorphisms by multiplex solid-phase minisequencing inmulti-capillary format using a detection system based on liquidcore waveguiding (<i>Paper IV</i>).</p><p>As these and other methods, as well as most applications inmolecular diagnostics and molecular biology, depend on thepolymerase chain reaction (PCR), an effort was made to enhancethe throughput of this technology and to minimize reactionvolumes and costs. For this, the concept of a dynamic reactorwas employed, instead of static systems where the reactionmixture is exposed to temperature cycles in a confined space. Acontinuous flow of small-volume reaction mixtures, separated byan immiscible hydrophobic carrier fluid such as aperfluorocarbon, is transported in a hydrophobic tube throughthree zones, which are kept at constant temperatures optimizedfor denaturation, annealing and elongation (<i>Paper V</i>). If combined with a technique for automatedsample loading and collection (<i>Chapter 7</i>), this method should allow veryhigh-throughput miniaturized DNA amplification. A samplehandling concept using hydrophilic anchors is proposed, whichshould also be useful for other miniaturized reactions andchemical processing.</p><p>Finally, some possible alternative methods are discussed aswell as future trends.</p><p><b>Keywords:</b>Amplification, anchor, array, assay,capillary, DGGE, disease, disorder, DNA, droplet, dynamicreactor, electrophoresis, fluorescence, fluorocarbon, gel,genetic alteration, genomics, genotyping, high-throughput,hydrophobic, liquid lid, miniaturized reaction, minisequencing,molecular diagnostics, mutation, PCR, polymerase chainreaction, screening, segmented flow, single nucleotidepolymorphism, SNP, SSCP, test.</p>

Lygus Hahn (Hemiptera: Miridae) are serious pests of economically important field, fruit, vegetable, and greenhouse crops in Canada. The release of European Peristenus Förster (Hymenoptera: Braconidae) in the USA has resulted in significant suppression of this pest and has renewed interest in the release of European Peristenus spp. in Canada. Prior to the release of exotic Peristenus spp., ecological host range studies need to be conducted to define their habitat and host associations. <p>These associations can be difficult to study using conventional methods. Morphological similarity of related parasitoids prevents species-level identification by dissection. Host rearing is time-consuming and can result in high levels of host and parasitoid mortality. To facilitate identification of immature Peristenus spp. in their hosts, a multiplex PCR assay was developed. This assay provided a specific and sensitive tool to screen individual insects for three parasitoid species simultaneously. <p>To validate the utility of the multiplex PCR assay in ecological host range studies, parasitism and parasitoid species composition obtained using conventional and molecular techniques were compared. Molecular methods compared favorably with conventional methods; however, more complete species composition information was available with the multiplex assay. To improve the quality of risk assessment studies and extract the most accurate ecological host range data, molecular methods were used to evaluate host-parasitoid associations in mirid populations collected in two ecoregions. Several new host-parasitoid associations were recorded for <i>P. digoneutis</i> and <i>P. relictus</i>, but parasitism of non-target mirids was low. <p>Parasitism of the target host collected from different plant species was evaluated to help clarify Peristenus host-plant associations. Despite the investigation of three different host plant species, no difference was observed in the parasitism level or parasitoid species composition in <i>L. rugulipennis</i>. The post-release utility of the multiplex assay was investigated in Canada, where Lygus parasitoids may have dispersed following release in the USA. To confirm establishment, samples were analyzed using the multiplex PCR assay, and P. digoneutis was detected for the first time in southern Ontario.

Lygus Hahn (Hemiptera: Miridae) are serious pests of economically important field, fruit, vegetable, and greenhouse crops in Canada. The release of European Peristenus Förster (Hymenoptera: Braconidae) in the USA has resulted in significant suppression of this pest and has renewed interest in the release of European Peristenus spp. in Canada. Prior to the release of exotic Peristenus spp., ecological host range studies need to be conducted to define their habitat and host associations. <p>These associations can be difficult to study using conventional methods. Morphological similarity of related parasitoids prevents species-level identification by dissection. Host rearing is time-consuming and can result in high levels of host and parasitoid mortality. To facilitate identification of immature Peristenus spp. in their hosts, a multiplex PCR assay was developed. This assay provided a specific and sensitive tool to screen individual insects for three parasitoid species simultaneously. <p>To validate the utility of the multiplex PCR assay in ecological host range studies, parasitism and parasitoid species composition obtained using conventional and molecular techniques were compared. Molecular methods compared favorably with conventional methods; however, more complete species composition information was available with the multiplex assay. To improve the quality of risk assessment studies and extract the most accurate ecological host range data, molecular methods were used to evaluate host-parasitoid associations in mirid populations collected in two ecoregions. Several new host-parasitoid associations were recorded for <i>P. digoneutis</i> and <i>P. relictus</i>, but parasitism of non-target mirids was low. <p>Parasitism of the target host collected from different plant species was evaluated to help clarify Peristenus host-plant associations. Despite the investigation of three different host plant species, no difference was observed in the parasitism level or parasitoid species composition in <i>L. rugulipennis</i>. The post-release utility of the multiplex assay was investigated in Canada, where Lygus parasitoids may have dispersed following release in the USA. To confirm establishment, samples were analyzed using the multiplex PCR assay, and P. digoneutis was detected for the first time in southern Ontario.

Were he alive today, would Louis Pasteur still champion culture methods he pioneered over 150 years ago for identifying bacterial pathogens? Or, might he suggest that new molecular techniques may prove a better way forward for quickly detecting the true microbial diversity of wounds? As modern clinicians faced with treating complex patients with diabetic foot infections (DFI), should we still request venerated and familiar culture and sensitivity methods, or is it time to ask for newer molecular tests, such as 16S rRNA gene sequencing? Or, are molecular techniques as yet too experimental, non-specific and expensive for current clinical use? While molecular techniques help us to identify more microorganisms from a DFI, can they tell us ‘who done it?', that is, which are the causative pathogens and which are merely colonizers? Furthermore, can molecular techniques provide clinically relevant, rapid information on the virulence of wound isolates and their antibiotic sensitivities? We herein review current knowledge on the microbiology of DFI, from standard culture methods to the current era of rapid and comprehensive ‘crime scene investigation' (CSI) techniques.

Piscicultura de água doce consiste na maior produção da aquicultura mundial e o cultivo de peixes tem crescido, devido a investimento, desenvolvimento de tecnologias e à contínua expansão de espécies cultivadas. Esse aumento gerou novas possibilidades de transmissão de vírus aquáticos, fatores limitantes à produção da aquicultura. Dentre as centenas de vírus conhecidos de peixes, um dos principais, que atualmente é de preocupação internacional, é o vírus da Viremia Primaveril da Carpa (SVCV), devido a sua importância socioeconômica considerando os países afetados e o comércio internacional de animais aquáticos e seus derivados, com base na saúde e medidas preventivas. SVC é uma doença aguda causada por um rhabdovírus do gênero Vesiculovirus, que é um vírus de ácido ribonucleico (RNA) fita simples linear de aproximadamente 11 quilobases (kb) com polaridade negativa e envelopado. Diversos ensaios diagnósticos podem ser utilizados para detectar SVCV, porém, consomem tempo e não são adaptados para diagnóstico a campo. A prevenção da disseminação do vírus é crucial; portanto, maior entendimento do vírus e de sua transmissão é necessário. No presente estudo, a padronização de uma RT-nestedPCR foi realizada, cujo limite de detecção foi de 8,62 × 10-2 cópias/reação, observado após o spiking de tecidos de peixes com diluições seriadas de um controle positivo sintético. O ensaio foi aplicado a 150 amostras teciduais provenientes de 146 peixes distintos. As amostras consistiam de fragmentos de fígado, rim e baço e foram submetidas à transcrição reversa (RT), seguida pela reação em cadeia de polimerase (PCR), em configuração nested. Duas (2) amostras foram positivas para o gene G de SVCV pela RT-nestedPCR e a identidade dos produtos obtidos foi confirmada por sequenciamento direto utilizando-se o método de Sanger. Na reconstrução filogenética, as sequências obtidas formaram clado único, separado dos demais genogrupos e mesmo de sequências derivadas de outros vesiculovírus encontrados no Brasil. Esses resultados determinam a primeira detecção e caracterização moleculares de SVCV no Brasil por RT-nestedPCR e sequenciamento nucleotídico, indicando a necessidade de adoção de medidas de biosseguridade mais restritivas na produção pesqueira nacional. / Fresh-water fish farming forms the largest portion of the world aquaculture production, and the harvest of these warm-water fish is increasing, due to investment, improvement of technologies and continued expansion of cultivated species. This increase has rendered new possibilities for the transmission of aquatic viruses, which remain limiting factors for aquaculture production. Among the hundreds of known viruses of fish, one of the main viruses that are currently of international concern is the Spring Viremia of Carp virus (SVCV) due to its socio-economic importance regarding affected countries and international trade of aquatic animals and their products, on the basis of health control and preventive measures. SVC is an acute disease, caused by a rhabdovirus, belonging to the Vesiculovirus genus, and is an enveloped virus, with a linear single-strained negative-sense ribonucleic acid (RNA) of approximately 11 kilobases (kb). Several diagnostic assays are available for the detection of SVCV, but they are time-consuming and not well adapted for field diagnosis. Prevention of spreading of the virus is crucial; therefore, more understanding of the virus and its transmission is required. In this study, standardization of a RT-nestedPCR was performed, and its detection limit was of 8.62 × 10-2 copies/reaction, observed after spiking fish tissue with serial dilutions of a synthetic positive control. The assay was applied to 150 tissue samples from 146 different fish. Samples consisted of liver, kidney and spleen fragments, and underwent reverse transcription (RT) followed by the polymerase chain reaction (PCR) of a nested configuration. Two (2) tissue samples were found positive for the G gene of SVCV by RT-nestedPCR and the identity of products was confirmed by direct sequencing using the Sanger method. By phylogenetic reconstruction, the obtained sequences formed a unique clade, separating them from the other known genogroups, and even from sequences derived from other vesiculoviruses found in Brazil. These results represent the first molecular detection and characterization of SVCV in Brazil by RT-nestedPCR and nucleotide sequencing, indicating the need to adopt more stringent biosecurity measures in national fish farming production.

Treatment success often depends on the availability of accurate and reliable diagnostic assays to guide clinical practitioners in their treatment choices. An optimal test must excel in specificity and sensitivity, and depending on the application area time, low-cost and simplicity are equally important. For instance, time is essential in infectious diagnostics but this is less important in non-invasive prenatal testing (NIPT). In NIPT, specificity and sensitivity are the most important parameters. In this thesis I describe the development of four different methods, all based on padlock probes and rolling circle amplification, intended for molecular diagnostics. Application areas range from infectious disease diagnostics to NIPT and oncology. The methods described have in common that they overcome certain limitations of currently available assays. This thesis includes two new assays targeting infectious agents: one assay specifically detecting a highly variable double stranded RNA virus and the second assay demonstrating a new format of antibiotic susceptibility testing, which is rapid and generally applicable to different pathogens. Furthermore, I describe the development of a method that uses methylation markers to enrich fetal DNA, accurately quantify chromosome ratios and thus, detecting trisomy 21 and 18. The fourth method described in this thesis uses gap-fill ligation of padlock probes to detect diagnostic relevant point mutations with high specificity in situ. The assays presented have the potential, after automation and successful validation and verification studies, to be implemented into clinical practice. Furthermore, these assays demonstrate the wide applicability of padlock probes which, due to their properties in regard to specificity and multiplexity, are useful tools for nucleic acid detection in vitro as well as in situ. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Piscicultura de água doce consiste na maior produção da aquicultura mundial e o cultivo de peixes tem crescido, devido a investimento, desenvolvimento de tecnologias e à contínua expansão de espécies cultivadas. Esse aumento gerou novas possibilidades de transmissão de vírus aquáticos, fatores limitantes à produção da aquicultura. Dentre as centenas de vírus conhecidos de peixes, um dos principais, que atualmente é de preocupação internacional, é o vírus da Viremia Primaveril da Carpa (SVCV), devido a sua importância socioeconômica considerando os países afetados e o comércio internacional de animais aquáticos e seus derivados, com base na saúde e medidas preventivas. SVC é uma doença aguda causada por um rhabdovírus do gênero Vesiculovirus, que é um vírus de ácido ribonucleico (RNA) fita simples linear de aproximadamente 11 quilobases (kb) com polaridade negativa e envelopado. Diversos ensaios diagnósticos podem ser utilizados para detectar SVCV, porém, consomem tempo e não são adaptados para diagnóstico a campo. A prevenção da disseminação do vírus é crucial; portanto, maior entendimento do vírus e de sua transmissão é necessário. No presente estudo, a padronização de uma RT-nestedPCR foi realizada, cujo limite de detecção foi de 8,62 × 10-2 cópias/reação, observado após o spiking de tecidos de peixes com diluições seriadas de um controle positivo sintético. O ensaio foi aplicado a 150 amostras teciduais provenientes de 146 peixes distintos. As amostras consistiam de fragmentos de fígado, rim e baço e foram submetidas à transcrição reversa (RT), seguida pela reação em cadeia de polimerase (PCR), em configuração nested. Duas (2) amostras foram positivas para o gene G de SVCV pela RT-nestedPCR e a identidade dos produtos obtidos foi confirmada por sequenciamento direto utilizando-se o método de Sanger. Na reconstrução filogenética, as sequências obtidas formaram clado único, separado dos demais genogrupos e mesmo de sequências derivadas de outros vesiculovírus encontrados no Brasil. Esses resultados determinam a primeira detecção e caracterização moleculares de SVCV no Brasil por RT-nestedPCR e sequenciamento nucleotídico, indicando a necessidade de adoção de medidas de biosseguridade mais restritivas na produção pesqueira nacional. / Fresh-water fish farming forms the largest portion of the world aquaculture production, and the harvest of these warm-water fish is increasing, due to investment, improvement of technologies and continued expansion of cultivated species. This increase has rendered new possibilities for the transmission of aquatic viruses, which remain limiting factors for aquaculture production. Among the hundreds of known viruses of fish, one of the main viruses that are currently of international concern is the Spring Viremia of Carp virus (SVCV) due to its socio-economic importance regarding affected countries and international trade of aquatic animals and their products, on the basis of health control and preventive measures. SVC is an acute disease, caused by a rhabdovirus, belonging to the Vesiculovirus genus, and is an enveloped virus, with a linear single-strained negative-sense ribonucleic acid (RNA) of approximately 11 kilobases (kb). Several diagnostic assays are available for the detection of SVCV, but they are time-consuming and not well adapted for field diagnosis. Prevention of spreading of the virus is crucial; therefore, more understanding of the virus and its transmission is required. In this study, standardization of a RT-nestedPCR was performed, and its detection limit was of 8.62 × 10-2 copies/reaction, observed after spiking fish tissue with serial dilutions of a synthetic positive control. The assay was applied to 150 tissue samples from 146 different fish. Samples consisted of liver, kidney and spleen fragments, and underwent reverse transcription (RT) followed by the polymerase chain reaction (PCR) of a nested configuration. Two (2) tissue samples were found positive for the G gene of SVCV by RT-nestedPCR and the identity of products was confirmed by direct sequencing using the Sanger method. By phylogenetic reconstruction, the obtained sequences formed a unique clade, separating them from the other known genogroups, and even from sequences derived from other vesiculoviruses found in Brazil. These results represent the first molecular detection and characterization of SVCV in Brazil by RT-nestedPCR and nucleotide sequencing, indicating the need to adopt more stringent biosecurity measures in national fish farming production.

Bird schistosomes of the genus Trichobilharzia are known as causative agents of hyper-immune skin reaction called cercarial dermatitis (swimmer's itch). They use pulmonary water snails from family Lymnaeidae as the intermediate host and mostly anatid birds as the definitive host. The first larva, miracidium, actively moves in water environment, penetrates the snail and develops to the mother sporocyst. Then the daughter sporocysts are formed and migrate to the hepatopancreas of the snail where the high number of cercariae is assexually produced. Cercariae leave the intermediate host, actively move in a water and penetrate the skin of definitive host. Within a host body they mature and lay eggs. Cercariae can penetrate also the mammalian skin, including human, where they are immediately eliminated by the immune system of the host, which is followed by inflammatory reaction. Until now, for humans, there is no effective method enabling to differ cercarial dermatitis from other hyper-immune skin reactions and for birds the reliable diagnostic method of trichobilharziasis is missing. The main aim of this thesis was to use the molecular methods for diagnostic of bird schistosomes infection in natural (ducks) and accidental hosts (mice, human). For optimization, the conventional PCR was used for detection...

Diagnostics are critical tools to assist in the identification of pathogens, the assessment of medical conditions, and helping to inform therapeutic decisions. Nevertheless, commonly used molecular diagnostics often require sophisticated instruments and skilled technicians, and therefore can only be done in centralized, well-equipped laboratories, which leads to long turnaround times, increased costs, and limited accessibility. These limitations have motivated the development of rapid, low-cost, decentralized diagnostics that are more widely accessible, affordable, and suitable for point-of-care applications. Synthetic biology, by creating rationally designed biological components that can sense disease markers, provides innovative and promising diagnostic solutions to achieve highly sensitive and specific detection for targets of interest, while at the same time being time- and cost-efficient, field-deployable, and shelf-stable. This dissertation focuses on the development of novel biosensing elements and their diagnostic applications. First, I introduce the methods for the computational design of riboregulators using automated algorithms. Followed by that, I describe the development, optimization, and applications of toehold-switch-based platforms for the detection of coccidioides, noroviruses, and severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). Next, I introduce the development of an ultra-specific riboregulator system termed single-nucleotide specific programmable riboregulators (SNIPRs) and their use for detecting different variants of concern of SARS-CoV-2. It is shown that riboregulators can be ideal solutions for various pathogen diagnostics with comparable accuracy and reduced cost. Lastly, I describe the use of peptide reporters derived from split protein systems to detect gene mutations. By incorporating peptide reporters into amplification primers, detection can be achieved by a quick isothermal amplification step and cell-free gene expression. Together, this research brings advancements in diagnostics based on riboregulators and cell-free systems that will increase the accessibility of these essential healthcare tools.