Дисертації: "Direct nucleic acid detection"

1

Lores, Lareo Pablo. "Nucleic acids and SNP detection via template-directed native chemical ligation and inductively coupled plasma mass spectrometry." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/20133.

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In den letzten Jahren gab es rasche Weiterentwicklungen auf dem Gebiet der Nukleinsäure-Erkennung. Von microRNA-Quantifizierung zur Untersuchung von Zelltods, --Teilung und -Regulation bis zur Bewertung genetischer Variabilität in Hinblick auf Krankheitsentstehung und -Behandlung: Die Analyse von Nukleinsäuren wird in der zukünftigen Medizin eine zentrale Rolle zukommen. Vor allem die Erkennung von SNPs als Hauptquelle der genetischen Vielfalt, aber aus Analysesicht auch eine der herausforderndsten Mutationen, stellt in dieser Hinsicht einen wesentlichen Aspekt dar. Methoden zur SNP-Erkennung müssen nicht nur sensibel, selektiv und stabil, sondern auch vielfältig sein und eine der wachsenden Analyseanzahl gerecht werdende hohe Verarbeitungsmenge bieten. Im Rahmendieser Arbeit wurde ein chemisches Prüfverfahren zur Erkennung von Nukleinsäuren und Einzelnukleotid-Polymorphismen (SNPs) entwickelt. Das Reaktionssystem zur Nukleinsäuren- Erkennung beruht hierbeiauf der Interaktion zweier modifizierter Peptid-Nukleinsäure (PNS) Oligonukleotiden. Das Erste beinhaltet einen C-terminalen Thioester (Donor-Sonde), die zweite einen N-terminalen Cysteinyl-Rest (Akzeptor-Sonde). Zusätzlich ist die Donor-Sonde durch einenmakrocyclischen Metall Chelatkomplex aus 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraessigsäure(DOTA) mit einem gebundenen lanthanoid-tag funktionalisiert. In die Akzeptor-Sonde wurde, zurReinigung mit magnetischen Streptavidin Partikeln, Biotin integriert. Der Ziel-DNA-Strang bringt beideSonden in räumliche Nähe zueinander und ermöglicht so eine chemische Reaktion. Das so gewonneneLigationsprodukt beinhaltet den Lanthanoid-Tag und Biotin, über welches das Produkt gereinigt wird,bevor die Detektion mittels ICP-MS erfolgt. Die Lanthanoid Konzentration dient als Indikator desLigationsprodukts welches wiederum den Reporter des Ziel-DNS-Strangs darstellt. Die, mithilfe diesesSystems erreichte, methodische Nachweisgrenze lag bei 29 pM mit einem RSD von 6,8% bei 50 pM(n=5). Zur Erkennung von SNPs wurde das Experiment mit einer Kombination zweier-Sets PNS Sonden mit unterschiedlichen Lanthanoid Tags durchgeführt. Das erste Set zielte auf die SNP beinhaltende Sequenz (Reportersystem) ab, während das zweite an eine benachbarte Sequenz (Kontrollsystem) binden sollte. Zur Erkennung der SNP wurden die Signale bei der Lanthanoide wurden ins Verhältnis gesetzt. Mithilfe dieses Verfahrens konnte durch Messung von sechs Lanthaniden bei einer Konzentration von 5 nM erfolgreich simultan zwischen den Allelen dreier SNPs unterschieden werden.
The field of nucleic acid detection has evolved swiftly in recent years. From quantification of micro RNA for the study of cell death, proliferation, and regulation, to the assessment of the influence of genetic variability towards disease development and treatment, the analysis of nucleic acids will play a central role in future medicine. In that regard, the detection of SNPs, as the primary source of genetic variability and the most challenging mutation from the analytical point of view, will be at the forefront of the discussion. Methods for the detection of SNPs not only require sensitivity, selectivity and robustness, but they should also allow multiplexing and offer high throughput in order to face the growing analysis demand In this work an assay for the detection of nucleic acids and single nucleotide polymorphisms (SNPs) was developed. The reaction system for the detection of nucleic acids is based on the interaction between two modified peptide nucleic acid (PNA) oligonucleotides. The first incorporated a C-terminal thioester (donor probe), and the second one a N-terminal cysteinyl residue (acceptor probe). In addition, the donor probe is functionalized with a metal-tag, which consist of a macrocyclic metal chelate complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with a chelated lanthanoide. A biotin tag for purification by streptavidin magnetic particles was incorporated in the acceptor probe. The target DNA strand brings together the reporter probes allowing the chemical reaction. The resulting ligation product contains the metal-tag and the biotin, which is used to purify the product before measurement in the ICP-MS system. The lanthanoid concentration is used as an indicator of the ligation product, which at the same time serves as reporter of the target template. The methodological limit of detection achieved with this system was 29 pM with RSD of 6.8% at 50 pM (n=5). Detection of SNPs was performed using a combination of two sets of PNA probes labeled with different lanthanoid metal tags. The first probe set targeted the sequence where the SNP was present (reporter probe system), while the second set of probes was designed to bind to a neighboring sequence (control probe system). The signals of both lanthanides were used to establish a ratio that allowed the detection of the SNP. This assay was successfully used to simultaneously differentiate between alleles of 3 SNPs by measuring six lanthanoids at 5 nM concentration.

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2

Chatwell, Nicola. "Nucleic acid approaches to toxin detection." Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.606582.

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PCR is commonly used for detecting contamination of foods by toxigenic bacteria. However, it is unknown whether it is suitable for detecting toxins in samples which are unlikely to contain bacterial cells, such as purified biological weapons. Quantitative real-time PCR assays were developed for amplification of the genes encoding Clostridium botulinum neurotoxins A to F, Staphylococcal enteroxin B (SEB), ricin, and C. perfringens alpha toxin. Botulinum neurotoxins, alpha toxin, ricin and V antigen from Yersinia pestis were purified at Dstl using methods including precipitation, ion exchange, FPLC, affinity chromatography and gel filtration. Additionally, toxin samples of unknown purity were purchased from a commercial supplier. Q-PCR analysis showed that DNA was present in crudely prepared toxin samples. However, the majority of purified or commercially produced toxins were not detectable by PCR. Therefore, it is unlikely that PCR will serve as a primary toxin detection method in future. Immuno-PCR was investigated as an alternative, more direct method of toxin detection. Several iterations of the method were investigated, each using a different way of labelling the secondary antibody with DNA. It was discovered that the way in which antibodies are labelled with DNA is crucial to the success of the method, as the DNA concentration must be optimised in order to fully take advantage of signal amplification without causing excessive background noise. In general terms immuno-PCR was demonstrated to offer increased sensitivity over conventional ELISA, once fully optimised, making it particularly useful for biological weapons analysis.

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3

Behrmann, Ole [Verfasser], and Gerald A. [Akademischer Betreuer] Urban. "Methods for rapid nucleic acid extraction and detection." Freiburg : Universität, 2021. http://d-nb.info/1227187289/34.

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4

Ferrier, David Christopher. "Nucleic acid detection using oligonucleotide cross-linked polymer composites." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28944.

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There has been much interest in recent years about the potential of microRNA as a new source of biomarkers for the diagnosis of disease. The delivery of new diagnostic tools based on this potential has been limited by shortcomings in current microRNA detection techniques. This thesis explores the development of a new method of microRNA detection through the incorporation of conductive particles into oligonucleotide-functionalised polymers to form oligonucleotide cross-linked polymer composites. Such composites could provide a simple, rapid, and low-cost means of microRNA detection that could be easily multiplexed, providing a valuable tool for point-of-care medical diagnostics. This work presents oligonucleotide-functionalised carbon/polyacrylamide composites which demonstrate a selective swelling response in the presence of analyte oligonucleotide sequences and for which the electrical conductivity decreases with swelling. The composites were synthesised via UV-initiated free-radical polymerisation of carbon/- monomer mixtures upon custom electrode devices, consisting of interdigitated platinum electrodes fabricated upon a silicon substrate. The optimal cross-linker density and carbon loading concentration were determined as well as the best means of dispersing the carbon particles within the polymer. Various types of carbon particles, with differing sizes and aspect ratios, were compared and their performances as conductive additives for polymer swelling transduction evaluated. The swelling behaviour of these composites was evaluated by analysing images of composite microdroplets as they swell. The electrical characteristics of the composites were determined by measuring either the two-terminal resistance or the complex impedance of composite microdroplets on the electrode devices. Alternating and direct current measurement techniques were compared to determine the best approach for the transduction of composite swelling. The volumetric and electrical responses of oligonucleotide-functionalised carbon/polyacrylamide composites were analysed in solutions of analyte oligonucleotide and non-complementary controls. It has been demonstrated that, using carbon nanopowder composites and a direct current two-terminal resistance measurement, it is possible to differentiate between analyte and control solutions to concentrations as low as 10 nM, with single-base precision, in less than three minutes. However, the inability to detect at concentrations below this value, difficulties in differentiating between different analyte concentrations and thermal instability mean that, in their current form, oligonucleotide cross-linked polymer composites are unsuitable for the detection of circulating microRNA at clinically relevant concentrations. Potential avenues of work to address these challenges are discussed. Also presented are collaborative results for oligonucleotide-responsive polymers functionalised with morpholino nucleic acid analogues, in what is believed to be the first example of such a material. These morpholino-functionalised polymers offer significant advantages, in terms of stability and sensitivity, over their nucleic acid equivalents for bio-responsive polymer applications.

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5

Gorgannezhad, Lena. "Advanced Technologies in Rapid and Multiplex Detection of Nucleic acid." Thesis, Griffith University, 2020. http://hdl.handle.net/10072/397045.

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Nucleic acids are key macromolecules of living organisms transferring genetic inheritance from one generation to the next. From how a living individual is created to how it interacts with external factors, all and all, can be found in the nucleic acid sequences inside every single cell of every organism. Therefore, the analysis of nucleic acids sequences is a critical capability for cancer and pathogen diagnoses, genotyping, and disease monitoring. To date, numerous methods have been used to detect both characterised and uncharacterised mutations and sequence variations. However, the detection of low amounts of mutant genes in the presence of high levels of wildtype sequences is still a challenge, and existing technologies has room for improvement. The classical approaches for nucleic acid detection and analyses mainly include DNA sequencing and the polymerase chain reaction (PCR). Although these methods with high analytical performance and reliability have facilitated the interrogation of the nucleic acids, some key obstacles such as the need of labelling, high costs for routine clinical use, slow turnaround time for giving results, the complexity of operation, and the inability to dually detect the genetic mutation in one step have limited their applications. To avoid drawbacks of the traditional approaches, a number of chip-based methods leverage electrochemical readouts or microfluidics to identify nucleic acids. However, there is still an unmet need for a less complex, rapid, low-cost, sensitive and accurate method to enable nucleic acid analysis even in resource-poor settings. The overall objective of this PhD thesis is to develop simple, inexpensive and accurate platforms for nucleic acid evaluation. To achieve the aforementioned goal, the first attempt was to develop a lab-on-a-chip platform for cancer diagnosis by detection of circulating tumor nucleic acids (ctNAs) in plasma samples of cancer patients. ctNAs are fragmented DNA released from cancerous cells and tumours into the bloodstream of patients with cancer. Tumour-specific (epi-)genetic alterations in ctNAs are assumed to reflect tumour burden and could be of high value for cancer diagnosis, prognosis, and management. In the first part of the thesis, I developed a new electrochemical assay for the detection of FGFR2:FAM76A fusion gene in ctNAs extracted from ovarian cancer patients. The assay was based on the high electrocatalytic activity of a new class of superparamagnetic graphene-loaded iron oxide nanoparticles. Electrochemical detection demonstrated a limit of detection (LOD) as low as 1.0 fM, high specificity and excellent reproducibility. In the second part of the thesis, I designed and developed a real-time and quantitative PCR system for microbial source tracking (MST) in water samples. MST is a DNA-based technology that enables water-quality managers to identify sources of faecal pollution in environmental waters. Most of the MST methodologies typically require specialized and costly equipment, elaborated and time-consuming operations as well as trained personnel. Here, a simple, low-cost, and sensitive platform was implemented on a microfluidic array chip. The array was successfully used for the real-time PCR-based multiplex detection of three human-associated MST markers (H8, Gen bac III, UidA). The PCR mixture was loaded into an array of channels in a single step utilising capillary filling without the need for liquid handling instruments. The array was then integrated with our custom-made thermal cycling and optical detection system. By employing the fabricated platform, the LOD of 71.8 DNA copies/μL was achieved for Gen bac III sequence. In summary, we introduced a sensitive, simple and economical real-time and quantitative PCR system for MST in water samples. In a further study, I investigated how a nucleic acid amplification setup can be miniaturised. To reach this goal, I utilised liquid marbles as an ideal biochemical microreactors for targeted amplification of the NAs. Liquid marbles are formed by encapsulating microscale volume of liquid with a thin layer of hydrophobic particles. Miniaturization of the nucleic acids (NAs) amplification process inside a liquid droplet provides several advantages upon routine methods, such as reducing reagents consumption and contamination possibility, easy handling of liquids, eliminating the usage of disposable plastic consumables for carrying out biochemical reactions. However, one of the major concerns in liquid marble applications is the high rate of evaporation through the porous walls during the thermal cycling step. To eliminate the evaporation, I used core-shell beads synthesized from a composite liquid marble as a NAs amplification micro reactor comprising two non-miscible liquid droplets forming a spherical shape and a coating of hydrophobic powder. The shell liquid was then polymerised into a solid after exposure to blue light, converting the liquid marble into a core-shell bead. Fabricated core-shell beads were extended to explore their potential as a versatile bioreactor for phylogrouping of the E.coli strains. In general, this platform provided easy manipulation and storage of sample, elimination of the evaporation, and sample protection from possible external contamination. Moreover, this simple and effective method presented a sensitive and inexpensive way to track NAs. In conclusion, this research endeavour presents a step forward towards the adaption of the selected group of tools and technologies, for the development of assays that can be applied as powerful alternatives to conventional tools used in molecular diagnostic. These technologies have the potential to revolutionise the NAs-based diagnostic approaches, by providing sensitive, rapid, accurate, and inexpensive platforms for point of care devices and in-field tests.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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6

Kershaw, David Michael. "Nanoparticle bound nucleic acid probes for DNA detection and gene inactivation." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7432/.

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In this project, a gold nanoparticle system has been developed that is able to detect SNP variations through a DNA based anthracene probe. A second probe is bound to the gold nanoparticle which allows the fluorescent output of the anthracene probe to be normalized. This allows the detection of SNP variations without the need for an initial reading, opening the possibility for using this system for cellular SNP identification. Through this work a new method for coating gold nanoparticles in oligonucleotides has been developed. In further work, the use of gold nanoparticles to deliver siRNA into cells and induce gene inactivation was investigated. Efforts to improve the knockdown efficiency of these siRNA-gold nanoparticles were made by integrating a second probe onto the nanoparticle surface, non-specific effects were observed upon addition of this second probe.

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7

Saeed, Ibrahim Q. "Optoelectronically active sensitisers for the selective detection of nucleic acid biomarkers." Thesis, Cardiff University, 2016. http://orca.cf.ac.uk/100885/.

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This thesis presents biophysical studies of new optoelecronically active DNA-binders. Chapter one gives a brief overview of the importance of DNA in medicine, of DNA structure and of the mode of interactions of small molecules with double-stranded DNA, including electrostatic, intercalation and groove interactions. Various examples of small-molecule binding to DNA are discussed. Additionally, this chapter briefly describes the biophysical techniques which can be exploited to quantify the interaction between small-molecules and duplex DNA. Chapter two describes the results of studies of the interactions of a group of 1,8-naphthalimide derivatives with double-stranded DNA using a variety of techniques viz. spectroscopy, calorimetry, viscosity and molecular docking studies. Additionally, this chapter also presents sequence selectivity studies of this group of compounds for specific sequences (dAdT)12●(dAdT)12 and (dGdC)12●(dGdC)12 through UV-visible spectroscopy. The 1,8-napthtalimide unit is shown to be a useful element for inducing DNA-binding. Chapter three describes studies of the interactions of a family of dendrimeric compounds with double-stranded DNA, again using spectroscopy, calorimetry, viscosity and molecular docking studies. Furthermore, this chapter includes sequence selectivity studies of this group of compounds for (dAdT)12●(dAdT)12 and (dGdC)12●(dGdC)12 via UV-visible spectroscopy. The charge and the length of the dendritic structures is shown to strongly affect nucleic acid affinities of this series of molecules. Chapter four describes the results of studies of the interactions of miscellaneous compounds with double-stranded DNA using variety of techniques viz. spectroscopy, calorimetry, viscosity and molecular docking studies. In addition, this chapter displays sequence selectivity studies of this group of compounds for specific sequences (dAdT)12●(dAdT)12 and (dGdC)12●(dGdC)12 via UV-visible spectroscopy. Chapter five gives an overview and general conclusions about the DNA binding studies presented in Chapters 2, 3 & 4 and finishes with suggestions for future work.

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8

O'Meara, Deirdre. "Molecular Tools for Nucleic Acid Analysis." Doctoral thesis, Stockholm : Tekniska högsk, 2001. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3220.

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9

Khater, Mohga Wagdy Yehia Mohamed. "Nanoparticle-based sensors for pathogen nucleic acid detection with interest for agriculture." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667373.

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La presente tesis describe el desarrollo de sensibles, bajo coste y portátiles métodos de sensado basados en nanomateriales aplicados en la detección de ADN de patógenos relacionados a plantas. El trabajo se presenta avances significativos en el campo de los biosensores para el diagnóstico de las enfermedades en las plantas. En el Capítulo I se da una visión general de las aplicaciones llevabas a cabo y mejoras aportadas por parte del uso de nanomateriales diferentes en el campo de los biosensores, y las nuevas aplicaciones en la detección de enfermedad de las plantas en lo punto de atención. En las secciones siguientes se presentan tres estrategias de sensado para la detección de secuencias de ADN específicas para el virus del cidro (citrus tristeza virus (CTV)), un virus modelo. Los sistemas están basados en las técnicas electroquímicas y ópticas. El Capítulo III se presenta el uso de electrodos serigrafiados de carbono (SPCEs) como plataforma para hibridación y detección del ADN mediante impedancia. Esta plataforma se adaptó mediante la disposición de nanopartículas de oro (AuNPs), con el fin de obtener una estrategia más simple, sin marcas, menos costosa y más rápida. Del mismo modo, en el Capítulo IV se centra en el desarrollo de nuevos métodos para la amplificación y la detección de ADN de CTV en SPCE modificados con AuNPs. Este biosensor opera en modo libre de marcas y con límite de detección (LOD) conseguido está en el rango de 1000 fg μL-1. Finalmente, en el Capítulo V se presenta la tercera plataforma utilizada, que fue basada en papel y tiene el formato de un inmunoensayo de flujo lateral (LFIA, del inglés lateral flow immunoassays). En esta plataforma, las nanopartículas de oro se usan como marcas para obtener señal de color rojo, con el fin de realizar nuevas aplicaciones en diagnóstico de plantas, más rápido y simple
This thesis aims at developing sensitive, affordable and portable biosensors based on nanomaterials for the determination of nucleic acid related to plant pathogens. The work strives to contribute to the keeping up in the advancements of biosensing systems relevant to plant infection diagnostics which would be an essential solution in the future to the issues of plant disease monitoring and food security. Following Chapter I, state-of-the-art on the latest trends in the development of advantageous biosensors based on both antibody and DNA receptors for early plant disease detection, as well as the use of different nanomaterials such as nanochannels and metallic nanoparticles for the development of innovative and sensitive biosensing systems for the detection of pathogens (i.e. bacteria and viruses) at the point-of-care is given. The next sections of this dissertation will describe three diagnostic biosensing strategies for the detection of citrus tristeza virus (CTV) related nucleic acid using electrical and optical transducing techniques. The electrical sensing of CTV through DNA hybridization based approach and the in situ amplified nucleic acid method will be achieved on carbon sensing substrate modified with gold nanoparticles, while paper-based sensors will be operated in lateral flow format for the gold nanoparticle-based optical detection of CTV. Furthermore, all aspects of the developed biosensing systems, from the bioassay and biosensor design to their development and optimization are presented in which will be organized in the following manner: Chapter III will present highly specific DNA hybridization sensor based on AuNP-modified SPCE employing label-free impedance for the detection of the CTV-related nucleic acid, together with dedicating emphasis to the study of electrodeposition time of AuNPs, whose precise particle size and shape will be required for the enhancement of DNA hybridization rate. A set of voltammetric studies of deposited AuNPs will be discussed. Particular attention will be paid for assembling the thiolated DNA probe as sensing layer for biosensor construction. The main sensor design aspects such as AuNPs size, probe DNA concentration and immobilization time together with DNA hybridization time will be optimized, in order to precisely select the best working conditions for this diagnostic platform. Chapter IV will cover the whole process undertaken for preparation of in situ nucleic acid amplification on gold nanoparticle-modified sensor for sensitive and quantitative detection of CTV. Plant disease (Citrus tristeza virus (CTV)) diagnostics was selected as relevant target for the demonstration of the proof-of-concept. This chapter will include two parts, the first one focuses on the design of RPA amplification assay, primers design, optimization of all essential bioassay aspects such as amplification temperature, volume and screening primers and finally the electrophoresis analysis for RPA products. The second part of this chapter will demonstrate label-free highly integrated in situ RPA amplification/detection approach at room temperature that takes advantage of the high sensitivity offered by gold nanoparticle-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection. Chapter V focuses on the application of isothermal nucleic acid amplification technology in simple lateral flow platform. The preparation of AuNP-based LFA for the highly sensitive direct detection of RPA amplified nucleic acid, the assembling of lateral flow step, the conjugation of AuNPs to the antibodies used for colorimetric detection, as well as the optimization of all working conditions and finally the analytical performance of the bioassay in LF will be explored. Moreover, aiming at truly achieving the point of care requirements of simple and affordable diagnostic technologies, the work here will present the possibility of amplifying nucleic acid without heat source and visual color detection. This approach would be of great potential as point of care diagnostics.

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10

Baloda, Meenu. "Lateral Flow Nucleic Acid Biosensor for the Detection of Sexually Transmitted Diseases." Diss., North Dakota State University, 2015. https://hdl.handle.net/10365/27596.

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Nucleic acid detection is of central importance for the diagnosis and treatment of genetic diseases, infectious agents, bio-warfare agents, and drug discovery. Nucleic acid testing for diseases is exclusively performed in laboratories using high-end instrumentation and personnel. However, this has developed the need for point of care diagnostics which can provide near-patient testing in a clinic, doctor’s office, or home. Such diagnostic tools can prove advantageous when rapid response is required or when suitable facilities are unavailable. Compared to equivalent methods used in laboratories, point of care testing is more affordable, as it eliminates the need for expensive instrumentation and skilled labor. One option involves the use of lateral flow assays. Pre-fabricated strips of dry reagents activated upon fluid application are already used in diagnostics, such as to ascertain pregnancy. Nucleic acid based detection assays on lateral flow offer several advantages over traditional microbiological detection methods. In this work we introduce a lateral flow biosensor that can combine the optical properties of nanoparticles (such as gold nanoparticles) with conventional immunoassay techniques to deliver a simple platform for rapid analysis of DNA with high sensitivity and selectivity. The quick 30 minute assay provides a platform to detect multiple nucleic acids with high efficiency achieved via chromatographic separation sandwich-type DNA hybridization reactions. Captured gold nanoparticles on the device can provide qualitative analysis by observing the color change to red and a semi-quantitative analysis via a strip reader. The biosensor was applied to the detection of human genomic DNA directly with high sensitivity and selectivity. The work was further expanded to detect Chlamydia trachomatis and Neisseria gonorrhoeae samples using nucleic acid amplification to generate large numbers of target copies. Improvements were made in the preparation of the biosensor to enable detection of Human Papilloma Virus Type-16. The clinical samples obtained were amplified using PCR for direct detection on the lateral flow biosensor without interference from other HPV types (e.g. HPV 18). The feasibility of the biosensor shows great potential for further development to assure its use in point of care diagnosis. The promising properties of the biosensor are reported in this dissertation.

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11

Stains, Cliff. "Methods for the Detection of Protein-Nucleic Acid and Protein-Protein Interactions." Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/194834.

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We describe the first general approach for the DNA templated reassembly of proteins, which we term SEquence-Enabled Reassembly or SEER. SEER makes use of dissected signaling domains which are each attached to separate, sequence specific DNA-binding proteins. Described herein is an embodiment of SEER in which DNA catalyzes the reassembly of the green fluorescent protein which leads to a direct fluorescence readout of the corresponding DNA sequence. This strategy has also been extended to the first direct method for the site specific detection of DNA methylation. This mCpG-SEER system is capable of discriminating between methylated versus nonmethylated DNA with a 40-fold increase in fluorescence signal.In a separate undertaking we tested the efficiency of disulfide bond formation within the context of the ribosome display in vitro selection methodology. We established conditions for the enrichment of a cyclic peptide, which is specific for Neutravidin, by 2 x 10^6-fold. Using the knowledge gained from the above experiments, we combined the rapid protein expression and folding benefits of cell-free translation systems with a sensitive split-luciferase reassembly assay to yield the most rapid method to date for the detection of protein-nucleic acid and protein-protein interactions. Furthermore, we have shown that these split-luciferase cell-free reassembly systems can be compartmentalized, allowing for future molecular evolution studies.Lastly, we have applied this rapid cell-free split-luciferase assay system to the direct detection of clinically relevant proteins. We have engineered a system for the rapid characterization of HIV-1 clades utilizing single-chain antibody specificities. We also demonstrate that this platform can be used to determine the relative amounts of HER2 expression in human breast cancer cells, using a homogeneous assay format in which cells and reagents are mixed and luminescence is monitored directly.We envision that the assay platforms described herein will find applications in the rapid detection of nucleic acid sequences, protein identities, and relative protein abundances in the laboratory and clinic.

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12

Mokany, Elisa Biotechnology &amp Biomolecular Sciences Faculty of Science UNSW. "The development of multi-component nucleic acid enzymes(MNAzymes)for the detection of analytes." Awarded by:University of New South Wales, 2007. http://handle.unsw.edu.au/1959.4/35210.

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13

Badran, Ahmed Hussein. "Split-Protein Systems for the Detection and Interrogation of Protein-Nucleic Acid Interactions." Thesis, The University of Arizona, 2010. http://hdl.handle.net/10150/146847.

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Cys2-His2 zinc fingers constitute one of the largest classes of DNA-binding domains in the human genome. The modularity of these domains has been recently exploited to design artificial zinc fingers, capable of targeting virtually any sequence. However, the resultant zinc fingers have had significantly high failure rate, owing to low binding affinity and selectivity. Despite much research on the topic, a proper understanding of all the factors involved in zinc finger selectivity, be they natural or artificial, has proved elusive. Here, we present a modification of our previously reported SEquence-Enabled Reassembly (SEER) methodology, allowing us to study zinc finger selectivity with base pair resolution. Using this modified strategy, we show that the natural 3-finger zinc finger Zif268 binding to its consensus site is highly dependent on the availability of specific base pairs, or 'hot spots,' and independent of mutations at adjacent positions. Additionally, we show that positional interdependence plays a large role in the selectivity of both Zif268 and the artificial 6-finger zinc finger Aart for their respective targets. We envision that this technique can be easily applied towards the interrogation of any DNA-binding domain in a high throughput and accurate manner.

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14

Thomas, Alistair Owen. "Detection of bacterial gene expression by a novel isothermic nucleic acid amplification technology." Thesis, University of Bath, 2004. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.410924.

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A novel probe-based technique called Signal-Mediated Amplification Reaction Technology (SMART) was optimised for detection of RNA targets in order to quantify gene expression. The SMART assay was used to quantify both 23 S rRNA in P. aeruginosa PAOl and gfpmuti mRNA in the plasmid-borne rpoSwgfpmvXi fusions P. aeruginosa SS429 and SS431. However, the assay was not sufficiently sensitive to detect gfpmvfo mRNA from the chromosomal rpoS::gfpmut3 fusion P. aeruginosa SS336. SDS-PAGE analysis of outer membrane proteins of P. aeruginosa PAOl revealed that cells grown in a reported iron-replete chemically defined medium CDMio were in fact limiting for iron. Modifications to the growth medium such as increased iron concentration, reduction in pH and the addition of citrate and ascorbate all failed to produce an iron-replete phenotype. This was achievable only when MOPSO buffer was replaced with phosphate buffer, indicating that by some unknown mechanism MOPSO can reduce iron availability in minimal media. The effects of nutrient limitation on rpoS expression in P. aeruginosa planktonic and biofilm culture were investigated using direct fluorescence measurement of rpoS::gfpmut3 chromosomal and plasmid fusions. In planktonic culture, nutrient-replete and magnesium-limited conditions resulted in an increase in rpoS expression whilst minimal levels of rpoS expression were seen in both iron-limited and glucose-limited conditions. Furthermore, minimal expression of rpoS was noted in P. aeruginosa biofilms in glucose, magnesium and iron-limited conditions.

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15

Gorbunova, Santa Maria. "Electrochemical characterization of carminic acid towards the use as an electrochemical molecular beacon for nucleic acid detection." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/52894.

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Worldwide, more than a million people die from tuberculosis (TB) every year. Although the disease is curable, treatment is complicated by multi-drug resistant and extensively drug-resistant TB strains. To detect TB and differentiate between its strains, a sensitive and specific point-of-care device is required. Previous studies show that carminic acid (CA), an anthraquinone derivative, is suitable as an electrochemical molecular beacon due to the ability to switch on and off its electrochemical activity on its dimerization. Characterization of the electrochemical activity of CA at low concentrations (1 μM to 1 mM) over a range of pH values was performed using methods such as cyclic voltammetry, square wave voltammetry and Koutecky-Levich analysis on a rotating disk electrode. CA species of different protonation, which are predominant at pH 1.1, pH 4.1, pH 6.6 and pH 10.5, were examined in more detail. All measurements were carried out on a glassy carbon electrode in phosphate buffer solution electrolyte. It was found that CA undergoes a diffusion limited two proton two electron redox reaction with an overall peak potential shift of 61 mV per pH unit. Electrochemical measurements of the fully protonated CA resulted in additional current peaks that were assigned to an adsorption process of a CA reduction product. Generally, CA has faster electron transfer kinetics in more acidic environment and no electrochemical activity was observed for the fully deprotonated CA species at pH 10.5. While SWV could be used for quantitative analysis of CA for the concentrations up to 1 mM, its redox current signal was determined not to be concentration dependent at high measurement frequencies. These frequencies can also be adjusted to be more sensitive towards either the redox peak potentials with sharper peaks at low frequencies or the electron transfer kinetics based on kinetic dependent peak currents at high frequencies. The limit of detection for CA at pH 7.0 was found to be as low as 10 nM when measured using 200 Hz SWV.
Science, Faculty of
Chemistry, Department of
Graduate

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16

Periyannan, Rajeswari Prem Kumar. "Droplet microfluidics for single cell and nucleic acid analysis." Doctoral thesis, KTH, Proteomik och nanobioteknologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-192668.

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Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening. The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes.

QC 20160926

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17

Zozulia, Oleksii [Verfasser], and Andriy [Gutachter] Mokhir. "Red light-triggered nucleic acid-templated reactions for detection of nucleic acids in live cells / Oleksii Zozulia ; Gutachter: Andriy Mokhir." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2017. http://d-nb.info/1135779791/34.

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18

Tomlinson, Jennifer A. "Nucleic acid-based methods for on-site detection of plant pathogens : approaches and applications." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12957/.

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The ability to perform nucleic acid-based detection of plant pathogens away from conventional laboratory facilities has the potential to be beneficial in situations where results are required very rapidly or where resources and access to laboratory equipment are limited. Methods for use in such situations must combine sensitivity and specificity with rapid and simple workflows. The aim of this project was to investigate aspects of on-site testing for plant pathogens by developing detection methods for a range of target species. Detection methods based on loop-mediated isothermal amplification (LAMP) exhibit characteristics which make them potentially suitable for on-site testing. LAMP-based methods were developed for detection of plant pathogens with three potential non-laboratory testing scenarios in mind: testing during plant health inspection (assays for Phytophthora ramorum, P. kernoviae and Guignardia citricarpa); testing to assess inoculum levels in the processing of plant products (an assay for Botrytis cinerea); and testing in under-resourced settings (assays for Cassava brown streak virus and Ugandan cassava brown streak virus). In developing these detection methods, attempts were made to address some of the specific requirements of potential end-users of the tests in each case. For testing in the context of inspection, a particular emphasis was placed on the need for simple, rapid methods for nucleic acid extraction. As well as investigating the use of rapid extraction methods in conjunction with LAMP, work was also carried out to investigate how on-site nucleic acid extraction using lateral flow devices could be integrated with current field and laboratory testing for P. ramorum.

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19

Xiao, Linlin. "Detection of Viable Foodborne Pathogens and Spoilage Microorganisms by Nucleic Acid Amplification Based Platforms." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1308284180.

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20

Derbyshire, Nicola. "Exploring the application of nucleic acid aptamers for detection of food contaminants and viruses." Thesis, University of Leeds, 2012. http://etheses.whiterose.ac.uk/4136/.

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Use of antibiotic, anti-fungal and anti-parasitic agents is required to protect from infections that can rapidly spread making entire crops unsuitable for human consumption or threaten the health of livestock. However, these agents can have toxic side effects on humans at high concentrations so their residual levels in food are regulated. Their levels are monitored through both random and routine surveillance checks that generate ~ 50 000 samples per year in the UK alone. This is not an easy task because many of these compounds are small molecules that require extensive sample extraction followed by analysis carried out by highly trained staff on sophisticated equipment, such as Liquid Chromatography–Tandem Mass Spectrometry. A simple, rapid and cheap method for screening these samples would therefore be beneficial in reducing the cost and time burdens of this monitoring. Nucleic acid aptamers are reagents that can be selected by a well-established in vitro protocol to bind to a wide range of target molecules. The flexibility of nucleic acids as primary capture ligands compared to antibodies means that they can be readily incorporated into rapid biosensors. Here I describe the selection and characterisation of aptamers against three classes of regulated residues, aminoglycoside (AMG) antibiotics, and the anti-fungal strobilurins and malachite green. The anti-AMG aptamers were toggle selected in an attempt to generate reagents able to recognise the entire class of drugs. Novel aptamers with such binding properties and Kd’s in the ~100 nM range were obtained. Some of these have been used in a simple and rapid biosensor based on aptamers physi-sorbed onto the surface of gold nanoparticles (GNPs). This proof of principle assay format showed promise in detecting aminoglycoside antibiotics at or below their permitted maximum residue levels. Next Generation DNA sequencing of the selected anti-AMG pools hints at the origin for the cross-reactivity. When further characterised, the previously selected anti-strobilurin aptamers appear to have specificity for their three selection targets. These aptamers were used to develop a high throughput screening method for aptamer target binding based on electrophoretic mobility shift assays with capillary electrophoresis. The assay has the potential to rapidly assess target binding of many sequences against many targets with limited user input. An anti-malachite green oxalate single stranded RNA aptamer that causes fluorescence on target binding has been previously described in the literature. I explored the potential of the single stranded DNA aptamers that I pseudo-toggle selected against malachite green oxalate and one of its metabolites to induce fluorescence on target binding. It appears that this phenomenon is unique to the single stranded RNA aptamer and malachite green oxalate In a separate attempt to illustrate the versatility of nucleic acid aptamers I have selected aptamers against the purified rabies virus glycoprotein (RVGP), which show both nano-molar affinity for the protein and also appear to have virus-neutralising properties. Rabies is endemic in many parts of the world and both pre- and post-exposure the major treatment relies on vaccination. Each year 100,000s of mice must be sacrificed following severe in vivo treatments in order to comply with the standard NIH-approved assay for vaccine potency. This level of in vivo testing is required because the anti-RVGP antibodies available are too variable in their response in ELISA assays to be a reliable indicator of potency. It is hoped that the selected aptamers will contribute to the development of such in vitro screens, thus reducing the need for animal testing.

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Hernández-Neuta, Iván. "Nucleic acid analysis tools : Novel technologies and biomedical applications." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-146334.

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Nucleic acids are fundamental molecules of living organisms functioning essentially as the molecular information carriers of life. From how an organism is built to how it responds to external conditions, all of it, can be found in the form of nucleic acid sequences inside every single cell of every life form on earth. Therefore, accessing these sequences provides key information regarding the molecular identity and functional state of any living organism, this is very useful for areas like biomedicine, where accessing and understanding these molecular signatures is the key to develop strategies to understand, treat and diagnose diseases. Decades of research and technological advancements have led to the development of a number of molecular tools and engineering technologies that allow accessing the information contained in the nucleic acids. This thesis provides a general overview of the tools and technologies available for nucleic acid analysis, and proposes an illustrative concept on how molecular tools and emergent technologies can be combined in a modular fashion to design methods for addressing different biomedical questions. The studies included in this thesis, are focused on the particular use of the molecular tools named: padlock and selector probes, rolling circle amplification, and fluorescence detection of single molecules in combination with microfluidics and portable microscopy. By using this combination, it became possible to design and demonstrate novel approaches for integrated nucleic acid analysis, inexpensive digital quantification, mobile-phone based diagnostics and the description of viral infections. These studies represent a step forward towards the adoption of the selected group of tools and technologies, for the design and building of methods that can be used as powerful alternatives to conventional tools used in molecular diagnostics and virology.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 1: Manuscript.

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22

Metcalf, Gavin Alan Dorman. "Fluorogenic Peptide Nucleic Acid probes for the detection of circulating microRNAs : applications to cancer diagnosis." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/45000.

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Cancer is a global burden with escalating incidence and mortality, often as a result of late diagnosis. Therefore, early detection, in order to improve prognosis and survival, remains a vital strategy for cancer management. A promising method for early diagnosis requires highly sensitive biomarkers and suitable technologies to detect them. Highly abundant in cells, microRNAs (miRNAs or miRs) play a key role as regulators of gene expression. A proportion of them are found circulating in biofluids, making them ideal non-invasive biomarkers for pathologies which aberrantly express miRNAs, such as cancer. Peptide Nucleic Acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridising to complementary nucleic acids with high affinity and specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesised to target specific miRNAs that were previously identified (and in some cases already validated) as potential diagnostic biomarkers for either Prostate Cancer (PCa) (miR-141 and miR-375) or Epithelial Ovarian Cancer (EOC) (miR-132). The sensing strategy is based on oligonucleotide-template reactions (OTRs) where only the miRNA of interest serves as a matrix to catalyse an otherwise highly unfavourable fluorogenic reaction. Validated in vitro using synthetic DNA and RNA oligonucleotides, these newly developed biosensors were then shown to detect endogenous concentrations of miRNA in human blood samples, without the need for any amplification step, and with minimal sample processing. This low-cost, quantitative and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost devices or assay kits. To investigate this further three platforms were explored where out PNA probes were compartmentalised into water-in-oil micro-droplets, embedded into permeable hydrogels or immobilised onto microplates. Low-cost and highly versatile, these platforms could pave the way for new point-of-care testing or public screening tools based on miRNA detection.

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23

Tsourkas, Andrew. "Development and optimization of dual FRET-molecular beacons for the detection and visualization of single-stranded nucleic acid targets." Diss., Georgia Institute of Technology, 2002. http://hdl.handle.net/1853/19256.

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24

Spencer, Sarah M. "Development of RNA Microchip for the Detection of Pathogens." Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/chemistry_diss/35.

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Detection of cellular messenger RNA is a useful diagnostic strategy for the detection of patho-gens. A rapid and sensitive method for on-site detection of specific pathogens would be of great use in a number of fields. For example, a simple and inexpensive method for the detection of harmful biological agents in train stations and airports is useful for national security. Rapid detection of pathogenic E. coli strains in food production would also be of great benefit in ensuring the safety and quality of our food supply. Here we present a method for the rapid de-tection of cellular mRNA. This system is based on the 3’-labeling approach in which targeted RNA is simultaneously extended and labeled with the use of biotin labeled-dNTPs and DNA po-lymerase on an immobilized nucleic acid probe. The biotin is subsequently converted to an enzymatic label, which produces a detectable chemiluminescent reaction in the presence of substrate. Detection time of this system is short (approximately 20 minutes) because there is no need for amplification by PCR, transcription, or fluorophore labeling. This novel methodology has been successfully demonstrated by selective detection of lac Z mRNA in a total RNA sample from E. coli.

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25

Grant, Paul Robert. "Development and application of nucleic acid amplification technology (NAT) for the detection of viruses in donated blood." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408734.

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26

Richardson, Kenneth James. "Use of nucleic acid probes and a nonradioactive labeling system for the detection of enteroviruses in water." Diss., The University of Arizona, 1989. http://hdl.handle.net/10150/184948.

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Enteroviruses affect a broad segment of the population throughout the world and have been suspected to play a major role in waterborne disease for quite some time. The presence of these viruses in drinking water supplies constitutes a major health risk to the population because of their low infectious dose. The monitoring and study of these viruses in the environment have been limited by the current standard detection methodologies. Nucleic acid probe hybridization is a new and effective approach for the study and detection of these viruses in the environment. An important step in the detection of viruses in concentrated water samples by nucleic acid probes is the isolation of the viral genome from the water sample for hybridization. Previously, a series of time consuming organic extract ions was used to isolate viral RNA. This study reports the development of an alternative method for the isolation and preservation of viral RNA in environmental samples. Briefly, the sample is heated in the presence of an RNase inhibitor, and then applied to a hybridization membrane. This procedure has greatly reduced the time and difficulty of the assay while maintaining sensitivity and increasing consistency. This study reports the development and modification of a nonradioactive labeling system for the detection of viruses in water. Nonradioactive labels such as biotin offer several advantages over radioactive labels including unlimited shelf life, reduced cost and time of assay, and elimination of the radiation hazard. However, radioactive labels are generally the more sensitive method of detection. By combining direct and indirect labeling strategies, the sensitivity of this nonradioactive assay has been increased ten-fold. This assay can detect as little as 100 plaque forming units of poliovirus, only one order of magnitude less sensitive than radiolabeled probes. This assay is also ten-fold less sensitive than radiolabeled probes for the detection of enteroviruses in water samples. Nonradioactive probes offer a safe, inexpensive alternative to radiolabeled probes and tissue culture for the detection of viruses in the environment when ultrasensitivity is not required.

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27

Sailis, Fiammetta. "Detection of miRNA by SMART technology." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28891.

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Aberrant expression of short non-coding micro RNAs (miRNA) in many human diseases, along with remarkable stability in physiological media, has made them attractive clinical biomarkers. In particular, miRNA-122 is substantially elevated in plasma of patients with established drug-induced liver injury and can also be used to identify early liver injury when current markers, such as alanine aminotransferase (ALT), still show normal levels. The development of a rapid test for miRNA-122 e.g. in drug poisoning would allow earlier and more sensitive clinical diagnosis of liver injury. Nucleic acids are traditionally analysed by polymerase chain reaction (PCR), which has a high degree of sensitivity but suffers from high cost and is prone to sample contamination. The aim of this project is to develop a PCR free method to directly detect miRNA- 122 in biological samples using SMART technology. The SMART technology takes advantage of dynamic chemistry for sequence specific recognition of nucleic acids using aldehyde-modified nucleobases (SMART nucleobases), and target-complementary peptide nucleic acid (PNA) probe containing an “abasic” position (so called modified PNA probe). In this study, this unique detection method was used in a fluorescent detection with the use of light up probes, which are probes with an environmental dye as nucleobase; a FRET system was also designed to allow the discrimination between perfect match target and mismatched one. The SMART technology was also transferred onto magnetic beads to develop an ELISA like assay allowing sensitive and rapid detection of single stranded DNA mimic of the miRNA-122. With its potential PCR free approach, this easily adapted platform promises to transform and expand routine clinical diagnostic testing and screening for circulating miRNAs.

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Kerr, Samantha Louise. "Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling." Thesis, Loughborough University, 2008. https://dspace.lboro.ac.uk/2134/4641.

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The application of ICP-MS to the fields of proteomics and genomics has arisen in part due to its ability to detect and quantify trace levels of S and P, which are major constituents in proteins and nucleic acids respectively. The development of collision/reaction cell technology and high resolution instruments has enabled these biologically important elements to be measured and quantified at the pg - ng ml-1 level. Despite these advances, the detection limits of P and S are still inferior compared to other elements. Oligonucleotides containing biotin functionality were labelled with Au nano-particles attached to a streptavidin protein to achieve site specific labelling, with 100% labelling efficiency. Each nano-particle contained ~86 Au atoms, resulting in an 882 fold signal enhancement for 24 base length oligonucleotides. However, this enhancement factor was only observed when one oligonucleotide bound to one nano-particle in a 1:1 ratio. Much lower Au labelling efficiencies and signal enhancements were observed when thiolated oligonucleotides were labelled with maleimide functionalised gold nano-particles. This was attributed to the extensive and difficult sample preparation steps that were required prior to labelling. The detection and quantification of adducts formed between DNA and the Pt anti-cancer drugs cisplatin and oxaliplatin were also investigated with ICP-MS. Acid digestion of the carbon based DNA matrix enabled Pt adducts to be quantified at low dose rates of 1 Pt atom per 1 500 000 nucleotides in ~12 μg DNA. Such sensitive mass spectrometric determinations could be employed in clinical tests to detect and quantify low level adducts formed in patients in-vivo. To complement ICP-MS analysis, electrospray ionisation linear ion trap mass spectrometry was employed to study the interaction of oxaliplatin with the four DNA nucleobases. Multiple stage mass spectrometry enabled detailed Pt-nucleobase adduct fragmentation pathways to be established. The method of DNA detection using P in conjunction with the collision cell, or cool plasma to form PO+ was also demonstrated and the limitations of the method, namely, polyatomic interferences and severe matrix effects were highlighted.

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Fredriksson, Simon. "Proximity Ligation : Transforming protein analysis into nucleic acid detection through proximity-dependent ligation of DNA sequence tagged protein-binders." Doctoral thesis, Uppsala University, Department of Genetics and Pathology, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2691.

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A novel technology for protein detection, proximity ligation, has been developed along with improved methods for in situ synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis.

Microarrays synthesized in situ using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.

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Zhang, Zhiling. "The structure and function of troponin T upon metal ion binding and the detection of nucleic acid sequence variations." Thesis, University of North Texas, 2005. https://digital.library.unt.edu/ark:/67531/metadc5565/.

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Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. This novel HYPA is expected to expand its applications in the analysis and screening of genetic diseases.

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Tang, Yeuk-nam Kennie. "A comparison of DIG nonradioactive with 32p radioactive nucleic acid labeling of Southern blot for the detection of alpha thalassaemia /." View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B32037661.

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32

Tang, Yeuk-nam Kennie, and 鄧若楠. "A comparison of DIG nonradioactive with 32p radioactive nucleic acid labeling of Southern blot for the detection of alpha thalassaemia." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B45010456.

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Silverman, Adam Phillip. "Part I: Detection of RNA in cells with quenched autoligation probes ; probing active site tightness in nucleic acid replication enzymes /." May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.

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Bernander, Sverker. "Detection and epidemiologic subtyping of Legionella pneumophila using DNA-based molecular methods /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-745-2.

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35

Nicolini, Ariana Marie, and Ariana Marie Nicolini. "Single-Step, Optical Biosensors for the Rapid and Sensitive Detection of Bacterial and Viral Pathogens." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/623158.

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This dissertation discusses the development of inexpensive, easy-to-use, and field-deployable diagnostic techniques and devices for the early detection of various pathogens, commonly found in clinical samples and contaminated food and water. Infectious diseases account for about 90% of world health problems, killing approximately 14 million people annually, the majority of which reside in developing countries. In 2012, the World Health Organization (WHO) published data on the top 10 causes of death across the globe. Although communicable disease is a prevalent cause of fatality, both low-income and high-income countries, pathogen species and transmission are very different. Nearly 60% of deaths in developing countries are caused by food, water, air or blood-borne pathogens. The most prevalent illnesses are diarrheal disease, malaria, and HIV/AIDS. By contrast, the leading causes of death in developed countries (heart disease, cancer, and stroke) are not communicable and are often preventable. However, there is an increasing need for the development of rapid and accurate methods for pathogen identification in clinical samples, due to the growing prevalence of antibiotic-resistant strains. Incorrect, or unneeded antibiotic therapies result in the evolution of extremely aggressive nosocomial (hospital-acquired) infections, such as methicillin- (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). The implementation of rapid, easy to use and cost-effective diagnostics will reduce the frequency of pathogen-related deaths in underdeveloped countries, and improve targeted antibiotic treatment in hospital settings, thus decreasing the potential development of more treatment-resistant "super bugs". This research includes novel techniques utilizing two major sensing modalities: serological (i.e. immunological), and nucleic acid amplification testing (NAATs). We first developed a highly sensitive (limit-of-detection = 100 CFU mL-1) particle immunoassay that takes advantage of elastic and inelastic light scatter phenomena, for optical detection of target antigens. This assay is performed upon a unique nanofibrous substrate that promotes multiplexing on a user-friendly platform. We then developed a novel technique, termed emulsion loop-mediated isothermal amplification (eLAMP), in which the target amplicon is detected in real-time, again utilizing light scattering detection and quantification. Both techniques require no sample pre-treatments, and can be combined with smartphone imaging for detection of targets in under 15 minutes. These methods have the potential to improve the speed and sensitivity of early pathogenic identification, thus leading to a reduction in preventative deaths and a decrease in global economic costs associated with infectious disease in clinical and other settings.

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36

Fun, Sze-tat. "Development of molecular diagnostic system for detection of hepatitis B virus in blood donations." Click to view the E-thesis via HKUTO, 2003. http://sunzi.lib.hku.hk/hkuto/record/B31971751.

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37

Pinto, Alessandro. "Real-time aptapcr: a novel approach exploiting nucleic acid aptamers for ultrasensitive detection of analytes for clinical diagnostic and in food analysis." Doctoral thesis, Universitat Rovira i Virgili, 2012. http://hdl.handle.net/10803/80744.

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The thesis aimed to develop and characterize a novel detection approach, which we termed aptaPCR exploiting nucleic acid aptamers as combined recognition and reporter biocomponents for the ultrasensitive detection of analytes. Nucleic acid aptamers are synthetic ligands selected from vast combinatorial libraries through a process referred to as SELEX – Systematic Evolution of Ligand By Exponential Enrichment. As compared to other natural and synthetic receptor, aptamers possess unique chemical and biochemical characteristics, such as: a well known chemistry, remarkable stability, an ability to be selected against virtually any target even in non-physiological conditions

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38

Nicholson, Wendy Jane. "The application of the polymerase chain reaction to the detection and characterization of human immunodeficiency virus and hepatitis B virus nucleic acid." Thesis, University of Edinburgh, 1994. http://hdl.handle.net/1842/21446.

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This thesis considers the application of PCR to the detection and characterisation of human immunodeficiency virus (HIV) nucleic acid and hepatitis B virus (HBV) DNA in clinical samples. Primers were selected from the po1 and env sequences for the detection of HIV-1 DNA, and amplifications of the envelope gene were used to demonstrate sequence variability. HIV-1 DNA was detected in all 32 patients (59 of the 61 PBMC samples tested). Viral DNA sequences were detected in nuclear extracts tested, 71% of cytoplasmic extracts, and in 50% of purified monocytic cells. Primers were selected to differentiate between HIV-1 DNA structural forms. Covalently closed circular unintegrated viral DNA (CUVD) with 1 or 2 LTR sequences was detected by amplifying across the junction site, and linear unintegrated viral DNA (LUVD) was detected using a ligation mediated PCR (L-PCR). CUVD was detected in 65% of patient samples and was associated with disease progression (0.02>P>0.01). The detection of LUVD was negative for all patient samples. HBV DNA was detected in patient serum samples using 4 specific primers in a nested PCR. The primers were selected to hybridize to the S-gene sequence of HBV using the nucleotide consensus sequence for 5 HBsAg subtypes (adrcg, adw, adyw, ayw, and awy₂). The presence of HBeAg in serum is indicative of infectivity. The detection of HBV DNA by PCR was compared with HBe status in 115 patient samples. All HBeAg positive and 25% of HBeAg negative samples were positive for HBV DNA. To determine the source of the viral DNA in these samples, the DNA extraction method was applied to free virus, and IgG and IgM complexed virus to establish the association of viral DNA with free virus and HBeAg.

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39

McCauley, Sean Matthew. "Innate Detection of HIV-1 in Myeloid Dendritic Cells." eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/993.

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Protective antiviral immune responses require priming of naïve T cells by dendritic cells (DCs) that have matured sufficiently to produce co-stimulatory cell surface molecules and cytokines. Although only low levels of productive HIV-1 infection are detected in ex vivo DCs following HIV-1 challenge, those few cells exhibit innate activation. Experimentally bypassing blocks to entry and replication leads to more efficient transduction of DCs and maturation as indicated by production of interferons and interferon stimulated genes. Furthermore, similar innate activation occurs upon transduction of macrophages or CD4+ T cells. However, the mechanism by which HIV-1 is detected to activate innate immune signaling is not clear. The purpose of this thesis is to incorporate my data and observations into the understanding of HIV-1 innate detection and attempt to resolve seemingly conflicting observations. Reverse transcription and genomic integration are necessary for innate activation implying the need de novo transcription. Coding sequences are unnecessary save for those cis-acting sequences necessary for the HIV-1 life cycle. CRM1 dependent, HIV-1 unspliced RNA export is essential for innate activation. As intact viral sequence is unnecessary for transcription and export, defective proviruses may contribute to systemic inflammation seen in chronically infected individuals. These insights, are hoped to aid in the production of qualitatively better anti-retroviral drugs as well as in the design a protective HIV vaccine.

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40

Weldhagen, Gerhard Frederick. "Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa." Thesis, University of Pretoria, 2004. http://hdl.handle.net/2263/29221.

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Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa tend to be geographically scattered, such as GES-2, which partially compromises the efficacy of imipenem. The G170N mutation, ascribed to a CC to AA base pair substitution on positions 493-494 of the blaGES-2 coding region, distinguishes this ESBL from blaGES-1 and the blaIBC-type genes, making it an ideal target for developing a novel sequence-specific, peptide nucleic acid (PNA)-based, multiplex-PCR detection method. Utilizing two primer pairs in conjunction with a PNA probe, this novel method delivered accurate identification of blaGES-2 compared to standard PCR and gene sequencing techniques, when tested against one hundred (n = 100) P. aeruginosa clinical isolates as well as previously published, well-described control strains. This method has the potential to be used in large-scale, cost-effective screening programmes for specific or geographically restricted ESBLs. To date, in addition to being only described in South Africa, GES-2 is notoriously difficult to identify in P. aeruginosa, using standard methodology. A real-time PCR method using the LightCycler™ was compared to a two-step nested-PCR assay for the detection of blaGES and blaIBC genes from one hundred P. aeruginosa clinical isolates collected over a four-year period from two teaching hospitals in Pretoria, South Africa. Real-time PCR amplification was monitored through hybridisation of fluorescently labelled probes followed by melting curve analysis to detect the relevant G170N mutation occurring in the omega loop region of blaGES-2. Nested-PCR products were subjected to automated DNA sequencing and compared to melting point (Tm) analyses results obtained from the LightCycler assay. Real time and nested-PCR assays detected a blaIBC gene product from 83 and 88 clinical isolates respectively, with the LightCycler thus exhibiting a sensitivity of 94.3% compared to the nested-PCR assay. Comparison of Tm and gene sequencing data however revealed 100% specificity for sequence specific detection of blaGES-2 with the LightCycler. One clinical isolate was found to harbour a blaGES-1 gene, making this the first report of this specific ESBL from South Africa. Selective antibiotic pressure has recently been implicated as a possible driving force behind point mutations observed in blaGES–type genes. This part of the study subjected two well-characterized clinical isolates with class 1 integron-borne blaGES-type genes to five days incubation in the presence of sub-inhibitory concentrations of 15 different antibiotics, including beta-lactams, aminoglycosides and quinolones. Restriction enzyme analysis and DNA sequencing of blaGES-1, blaGES-2 and their immediate upstream genetic environments failed to demonstrate any changes compared to non-exposed controls. Short-term exposure to a sub-inhibitory level of a single antimicrobial agent is thus unlikely to select significant mutations in these beta-lactamase genes or their regulatory mechanisms.
Thesis (PhD (Medical Microbiology))--University of Pretoria, 2006.
Medical Microbiology
unrestricted

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41

Porter, Jason Robert. "SPLIT-PROTEIN REASSEMBLY METHODS FOR THE DETECTION AND INTERROGATION OF BIOMOLECULAR INTERACTIONS AND MODULATORS THEREOF." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/194359.

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The interactions between protein-protein, protein-nucleic acid, and protein-small molecules are central to biological processes and are key for the design of new therapeutics. Rapid and easy to implement methodologies are needed that enable the interrogation of these interactions in a complex cellular context. Towards this goal, I have utilized the concept of split-protein reassembly, also called protein complementation, for the creation of a variety of sensor architectures that enable the interrogation of protein-nucleic acid, protein-protein, and protein-small molecule interactions. Utilizing the enzymatic split-reporter β-lactamase and existing zinc finger design strategies we applied our recently developed technology termed SEquence-Enabled Reassembly (SEER) towards the creation of a sensor capable of the specific detection of the CryIA transgene. Additionally, the split β-lactamase reporter was utilized for the sitespecific determination of DNA methylation at cytosine residues that is involved in epigenetic regulation. This method, dubbed mCpG-SEER, enabled the direct detection of femtomole levels of dsDNA methylation in sequence specific manner. In a separate endeavor, we have developed and optimized the first cell-free split-reporter systems for GFP, split β-lactamase, and firefly luciferase for the successful dsDNA-dependent reassembly of the various reporters. Our cell free in vitro translation systems eliminates previous bottlenecks encountered in split-reporter technologies such as laborious transfection/cell culture or protein purification. Capitalizing on the ease of use and speed afforded by this new technology we describe the sensitive detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions and inhibitors thereof. In a related area, we have applied this rapid cell-free split-firefly luciferase assay to the specific interrogation of a large class of helix-receptor protein-protein interactions. We have built a panel consisting of the clinically relevant Bcl-2 family of proteins, hDM2, hDM4, and p53 and interrogated the specificity of helix-receptor interactions as well as the specificity of peptide and small-molecule inhibitors of these interactions. Finally, we describe the further applications of our cell-free technology to the development of a large number of general split-firefly luciferase sensors for the detection of ssRNA sequences, the detection of native proteins, the evaluation of protease activity, and interrogation of enzyme-inhibitor interactions. The new methodologies provided in this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.

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42

Lehnus, Massimiliano. "Bio-BCA (Bio-Barcode Cascade Amplification) : development of a photosensitive, DNA-based exponential amplification platform technology for the detection of nucleic acid biomarkers." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277915.

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43

Seitz, Oliver [Gutachter], Michael W. [Gutachter] Linscheid, and Ilko [Gutachter] Bald. "Nucleic acids and SNP detection via template-directed native chemical ligation and inductively coupled plasma mass spectrometry / Gutachter: Oliver Seitz, Michael W. Linscheid, Ilko Bald." Berlin : Humboldt-Universität zu Berlin, 2019. http://d-nb.info/119993061X/34.

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44

Mantha, Stacey. "Direct detection and rapid identification of clinically significant mycobacteria from Bactec 12B medium and acid-fast bacilli positive sputum specimens." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0015/MQ54472.pdf.

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45

Lam, Yiu-pong, and 林耀邦. "Performance evaluation of the automated NucliSens easyMAG and Qiagen EZ1 Advanced XL nucleic acid extraction platform for detection of RNAand DNA viruses in clinical samples." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46448020.

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46

Fun, Sze-tat, and 范思達. "Development of molecular diagnostic system for detection of hepatitis B virus in blood donations." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31971751.

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47

Faltin, Bernd [Verfasser], and Roland [Akademischer Betreuer] Zengerle. "Mediator Probe PCR: a novel assay principle for universal real-time detection of nucleic acid amplification = Mediator Probe PCR: ein neuartiger Ansatz zur universellen Echtzeit-Detektion von Nukleinsäuren." Freiburg : Universität, 2013. http://d-nb.info/1123477000/34.

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48

Ceylan, Koydemir Hatice. "Mems Based Electrochemical Dna Sensor To Detect Methicillin Resistant Staphylococcus Aureus And Vancomycin Resistant Enterococcus Species." Phd thesis, METU, 2013. http://etd.lib.metu.edu.tr/upload/12615402/index.pdf.

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Methicillin Resistant Staphylococcus aureus (MRSA) is one of the most important threats of nosocomial infections in many regions of the world and Vancomycin Resistant Enterococcus (VRE) is an emerging pathogen that develops full resistance against third-generation glycopeptide antibiotics. Conventional methods for identification of MRSA and VRE generally depend on culturing, which requires incubation of biological samples at least 24-72 hours to get accurate results. These methods are time consuming and necessitate optical devices and experts for evaluation of the results. On the other hand, early diagnosis and initiation of appropriate treatment are necessary to decrease morbidity and mortality rates. Thus, new diagnostic systems are essential for rapid and accurate detection of biological analytes at the point of care. This study presents design, fabrication, and implementation of MEMS based micro electrochemical sensor (µ
ECS) to detect the methicillin resistance in Staphylococcus aureus and vancomycin resistance in Enterococcus species. To the best of our knowledge, the developed sensor is the first µ
ECS which utilizes on-chip reference (Ag), working (Au), and counter (Pt) electrodes together with a microchannel to detect MRSA and VRE. The characterization of the designed sensor was achieved analyzing the interactions of the buffer solutions and solvents with the electrodes and Parylene C film layer by using optical and electrochemical methods. Specific parts of genes that are indicators of antimicrobial resistances were used in order to detect the resistances with high selectivity and sensitivity. Thus, synthetic DNA and bacterial PCR product were used as target probes in redox marker based detection and enzyme based detection, respectively. In order to enhance the hybridization, folding structures of the capture probe were investigated by using mfold Web Server. In redox marker based detection, the hybridization of DNA was indirectly detected by using Hoechst 33258 as redox marker with differential pulse voltammetry. The cross reactivity of the tests were performed by using different target probes of femA genes of S. aureus and S. epidermis, which are the major genes detected in methicillin detection assays. Consequently, amplification of signal by using horseradish peroxidase and TMB/H2O2 as substrate was achieved in order to enhance detection sensitivity. The sensor could detect 0.01 nM 23-mer specific part of mecA gene with redox marker based detection and 10 times diluted PCR product with enzyme-based detection in about six hours including the steps of sample preparation from whole blood. This sensor with its compatibility to MEMS fabrication processes and IC technology has a promising potential for a hand-held device for POC through the integration of micropotentiostat.

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49

VERMA, SUJIT KUMAR [Verfasser], Sebastian [Akademischer Betreuer] Springer, Sebastian [Gutachter] Springer, Michael [Gutachter] Köhler, Mathias [Gutachter] Winterhalter, and Werner M. [Gutachter] Nau. "Polyelectrolyte Microcapsules: A versatile and sensitive tool for the detection of protein and nucleic-acid analytes / SUJIT KUMAR VERMA ; Gutachter: SEBASTIAN SPRINGER, MICHAEL KÖHLER, MATHIAS WINTERHALTER, WERNER M. NAU ; Betreuer: SEBASTIAN SPRINGER." Bremen : IRC-Library, Information Resource Center der Jacobs University Bremen, 2017. http://d-nb.info/114810397X/34.

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50

Fischbach, Melanie. "Haarnadelförmige PNA-Peptid-Konjugate." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17305.

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Das Entwicklungsstadium bestimmter Krankheiten ist eng mit der Konzentration diverser Proteine in biologischen Proben verknüpft. Eine sensitive Detektion dieser sogenannten Biomarker kann somit maßgeblich zu einer frühzeitigen Diagnose beitragen. In der vorliegenden Arbeit wurden strukturierte, fluorogene Sonden entwickelt, die die Möglichkeit bieten in einem homogenen Verfahren Zielproteine sensitiv, direkt und in Echtzeit nachzuweisen. Die peptidische Erkennungssequenz für das Zielprotein wurde dabei von zwei zueinander komplementären PNA-Segmenten flankiert. Die Sonden besaßen dadurch eine haarnadelförmige Anordnung, die kontrolliert eingebaute Reporter in eine enge Proximität zwang und ein minimales Hintergrundsignal im ungebundenen Zustand gewährleistete. Durch die Wechselwirkung mit dem Zielprotein erfolgte eine Reorganisation der Sondenstruktur, die fluoreszenzspektroskopisch verfolgt werden konnte. Für den Einbau der fluorogenen Einheiten wurden verschiedene Strategien entwickelt und die resultierenden Architekturen bzgl. ihres Einsatzes als sensitives Detektionssystem validiert. Als Zielproteine wurden die intrazellulären SH2-Domänen der Src- und Lck-Kinase sowie die extrazelluläre Matrix-Metalloprotease MMP-7, ein proteolytischer Biomarker für Krebs, untersucht. Besonders die neuartigen In-Stem Hairpin Peptide Beacons (IS-HPBs), bei denen fluorogene Pseudonukleobasen in die PNA-Stammregion eingebaut wurden, zeichneten sich als sensitive Proteasereporter mit einer bis zu 50-fachen Signalverstärkung aus. Mit einem excimerbasierten IS-HPB und einer zeitaufgelösten Fluoreszenzmethode konnte die direkte Detektion von MMP-7 bei einer kritischen Konzentration von 1 nM im humanen Blutserum erreicht werden. Eine mögliche Anwendbarkeit in der medizinischen Diagnostik wurde somit bekräftigt. Weiterhin wurden erste Hinweise mithilfe thermodynamischer Untersuchungen erhalten, dass die Strukturierung einer peptidischen Sonde zu einer erhöhten Selektivität beiträgt.
The developmental stage of certain diseases is closely linked to the concentration of various proteins in biological samples. A sensitive detection of these so-called biomarkers can thus significantly contribute to an early diagnosis. In the present work, structured, fluorogenic probes were developed that offer the possibility of a sensitive, direct and in real-time detection of target proteins in a homogeneous process. The peptidic recognition sequence for the target protein was thereby flanked by two self-complementary PNA segments. As a result, the probes possessed a hairpin-type arrangement, in which suitable appended labels are forced into close proximity and guaranteed a minimal background signal in the unbound state. By interacting with the target protein a reorganization of the probe structure occured, which could be followed by fluorescence spectroscopy. To embed the fluorogenic units different approaches were developed and the resulting architectures were validated relating to their use as a sensitive detection system. As target proteins the intracellular SH2-domains of the Src and Lck kinase and the extracellular matrix metalloprotease MMP-7, a proteolytic biomarker for cancer, were investigated. In particular, the new In-Stem Hairpin Peptide Beacons (IS-HPBs), in which fluorogenic pseudo nucleic acids were incorporated into the PNA-stem region, proved as sensitive protease reporters with an up to 50-fold signal amplification. By using an excimer-signaling IS-HPB and a time-resolved fluorescence method the direct detection of MMP-7 with a critical concentration of 1 nM within complex human blood serum was achieved. A possible application in medical diagnostics was thus confirmed. Furthermore, initial indications were obtained using thermodynamic studies that the structure of a peptide-based probe contributes to increased selectivity.

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