Дисертації: "Nucleic-acid Amplification and Quantification"

1

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At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 1: Manuscript.

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2

Lee, Dong-Hun. "Nucleic acid amplification testing for screening of individual blood units." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2007. http://dare.uva.nl/document/48208.

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3

Daher, Rana. "Recombinase polymerase amplification technology : Assessment for nucleic acid-based acid-based point-of-care diagnostics." Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/26269.

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Cette thèse de doctorat porte dans l’ensemble une étude approfondie sur une technologie émergente pour l’amplification isotherme des acides nucléiques appelée recombinase polymerase amplification (RPA). L’introduction porte une description détaillée sur la RPA. Cette revue de littérature documente et discute les diverses applications de la RPA en soulignant les connaissances actuelles concernant les applications diagnostiques. Malgré la composition complexe de la RPA (6 à 7 protéines dans le même mélange réactionnel), cette dernière s’avère une technologie rapide (générant des résultats < 20 min), spécifique et sensible (détection de l’ordre de quelques copies de génome), et largement appliquée dans différentes disciplines. Ces avantages nous permettent de croire que la RPA possède la flexibilité nécessaire pour être utilisée comme outil de diagnostic rapide des maladies infectieuses en réduisant le temps d’obtention des résultats à moins d’une heure au lieu de 2 à 3 jours avec les tests de cultures standards. En conséquence, il sera possible d’intégrer la RPA dans des plateformes microfluidiques ou laboratoire sur puce qui permettent la préparation d’échantillons, l’amplification et la détection des acides nucléiques des microbes causant des infections. En premier lieu, les travaux de cette thèse ont généré des lignes directrices additionnelles pour la conception des amorces/sondes RPA. En second lieu, nos travaux ont permis de développer un essai diagnostic RPA pour la détection des streptocoques du groupe B, responsables de la septicémie et la méningite chez les nouveau-nés. Cet essai fut le premier à évaluer la performance de la RPA avec des échantillons cliniques humains. Ce test diagnostic RPA a été comparé à une méthode de référence, la réaction en chaîne par polymérase (PCR). Cette démonstration sur des échantillons cliniques nous à inciter à pousser notre étude pour réaliser le dernier objectif de ce projet qui consistait à automatiser la RPA par intégration dans un système microfluidique miniaturisé centripète. Une collaboration avec des experts en génies et en matériaux a permis de générer un dispositif microfluidique appelé blade ainsi de l’instrument impliqué dans l’opération des différentes tâches mécanistiques. Ces résultats préliminaires suggèrent qu’il sera important d’offrir un système automatisé complet applicable au chevet du patient. Par conséquence, il sera possible d’exécuter une analyse complète des agents infectieux en moins d’une heure sans le besoin des procédures complexes de préparation et de transport des échantillons cliniques ni le recours à du personnel qualifié.
This dissertation consists of an exhaustive study on an emerging technology for isothermal amplification of nucleic acids called recombinase polymerase amplification (RPA). The introduction of this thesis is a detailed description of the RPA. This review documents and discusses the various applications of this technology by pointing to the current knowledge about RPA for diagnostic applications. Despite the complex composition of RPA (6 to 7 proteins in the same reaction mixture), the latter was shown to be rapid (generating results in < 20 min), specific and sensitive (detecting few target genome copies), and applied widely in different fields. Based on these advantages, we assume that RPA has a flexibility allowing it to be used for the rapid diagnosis of infectious diseases thus reducing time-to-result to less than an hour. Consequently, it will be possible to integrate RPA in microfluidic platforms providing a lab-on chip system. The first part of this doctoral project generated additional guidelines for RPA primers/probes design to develop specific RPA diagnostic assays. Second, we developed an RPA diagnostic test for the detection of group B streptococci, responsible for sepsis and meningitis in newborns. This assay was the first to evaluate RPA with human clinical samples. This diagnostic test was compared to a reference method, the polymerase chain reaction (PCR). This demonstration with clinical samples served to carry out the final objective of this project that was to automate RPA in a miniaturized microfluidic centripetal system. Collaboration with engineers and experts in materials has generated the microfluidic device called "blade" and the instrument involved in the operation of various mechanistic tasks. These preliminary results suggested that it will be important to provide an automated system applicable at bedside. Consequently, it will be possible to perform a complete analysis of infectious agents in less than an hour without the need for complex procedures for the preparation and transport of clinical specimens or the assistance of qualified personnel.

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Matinyenya, Brian. "Novel and newer nucleic acid amplification tests for the diagnosis of TB." Master's thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/20680.

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Background: Current tools for TB diagnosis have suboptimal accuracy, perform poorly in diagnosing extra-pulmonary TB, and are not point of care; hence results have a slow turn-around time. Objective: This project evaluated the diagnostic accuracy of the promising novel loop mediated isothermal amplification (LAMP) assay on sputum, and that of the semi-automated Xpert MTB/RIF (Xpert) test on non-sputum specimens (bronchoalveolar lavage fluid [BALF], tracheal aspirates, and cerebrospinal fluid [CSF]) from South African patients with suspected TB (the accuracy of Xpert using these fluids was unknown at the time this work was performed). Methodology: Biological samples (sputum, tracheal aspirates, BALF, or CSF) were collected from patients with suspected TB. Liquid culture served as the reference standard for the diagnosis of definite TB. Accuracy was evaluated according to HIV and smear microscopy status, where appropriate. The relationship between test performance and bacterial load (culture time-to-positivity [TTP]) was also compared. For the evaluation of LAMP, 2 spot sputa of approximately 4 ml were collected from 301 patients (60 μl of sputum was used for the assay). For the evaluation of Xpert on BALF, 152 patients who were sputum scarce or smear-negative were recruited (1 ml of the BALF aliquot or a re-suspended pellet from 10 ml BALF was used). For the evaluation of Xpert on tracheal aspirates, 120 tracheal aspirates from patients enrolled in the intensive care unit (ICU) were tested. For the evaluation of Xpert on CSF, 235 patients with suspected TBM had a lumbar puncture with 1 ml of CSF or where available a re-suspended pellet from 3 ml of CSF evaluated using Xpert.

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Syed, Shahida Nina. "Electrochemical control of reversible DNA hybridisation : for future use in nucleic acid amplification." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9617.

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Denaturation and renaturation is indispensable for the biological function of nucleic acids in many cellular processes, such as for example transcription for the synthesis of RNA and DNA replication during cell division. However, the reversible hybridisation of complementary nucleic acids is equally crucial in nearly all molecular biology technologies, ranging from nucleic acid amplification technologies, such as the polymerase chain reaction, and DNA biosensors to next generation sequencing. For nucleic acid amplification technologies, controlled DNA denaturation and renaturation is particularly essential and achieved by cycling elevated temperatures. Although this is by far the most commonly used method, the management of rapid temperature changes requires bulky instrumentation and intense power supply. These factors so far precluded the development of true point-of-care tests for molecular diagnostics. This Thesis explored the possibility of using electrochemical means to control reversible DNA hybridisation by using electroactive intercalators. First, fluorescence-based melting curve analysis was employed to gain an in depth understanding of the reversible process of DNA hybridisation. Fundamental properties, such as stability of the double helix, were investigated by studying the effect of common denaturing agents, such as formamide and urea, pH and monovalent salt concentration. Thereafter, four different electroactive intercalators and their effect on the thermodynamic stability of duplex DNA were screened. The intercalators investigated were methylene blue, thionine, daunomycin and adriamycin. Absorbance-based melting curve analysis revealed a significant increase of the melting temperature of duplex DNA in the presence of oxidised daunomycin. This was not observed in the presence of chemically reduced daunomycin, which confirmed the hypothesis that switching of the redox-state of daunomycin altered its properties from DNA binding to non-binding. Accordingly this altered the thermodynamic stability of duplex DNA. The difference in the stability of duplex DNA, as a direct result of the redox-state of daunomycin, was exploited to drive cyclic electrochemically controlled DNA denaturation and renaturation under isothermal conditions. This proof-of-principle was demonstrated using complementary synthetic 20mer and 40mer DNA oligonucleotides. Analysis with in situ UV–vis and circular dichroism spectroelectrochemistry, as two independent techniques, indicated that up to 80 % of the duplex DNA was reversibly hybridised. Five cycles of DNA denaturation and renaturation were achieved and gel electrophoresis as well as NMR showed no degradation of DNA or daunomycin. As no extreme conditions were implicated, no covalent modification of DNA was required and isothermal conditions were kept, this finding has great potential to simplify future developments of miniaturised and portable bioanalytical systems for nucleic acid-based molecular diagnostics.

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6

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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7

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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8

CORAL, LUCIA. "HIGH-RESOLUTION NUCLEIC ACID ANALYSIS WITH A DNA NANOTECHNOLOGY APPROACH." Doctoral thesis, Università degli Studi di Trieste, 2017. http://hdl.handle.net/11368/2908115.

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The goal of my research program is to develop a DNA-based nanosensor for nucleic acids analysis. I plan to use DNA Origami nanostructures that are formed by a-few-thousand-nucleotides-long, circular, single stranded (ss)DNA “scaffold” folded to form a specific shape by the action of a few hundreds of short (approx. 30 nucleotides) ssDNA “staples”, which hybridize over non-consecutive regions of the scaffold. Staples can be incorporated within the structure with well-defined stoichiometry and some of them can be designed to serve as highly-specific receptor for short nucleic acids sequences. I plan to introduce a restriction site within staples adjacent to such probes to permit their steric protection from enzymatic degradation as a consequence of a probe-target recognition event in their vicinity. The restriction reaction, therefore, “writes” the amount of target molecules captured on the nanosensors by permanently modifying certain target-specific staples within the DNA nanostructure. In turn, the amount of such modified staples is associated with the amount of target molecules captured on the nanosensor surface from the solution, and can be subsequently analyzed with standard DNA quantification techniques such as quantitative PCR (qPCR), or high-throughput DNA sequencing. This research plan is based on recent results obtained in our laboratory showing that, in self-assembled DNA structures, restriction enzymatic reactions are steric-regulated in a step-wise fashion. Therefore, one goal of this PhD thesis is the development of a quantitative assay to evaluate the efficiency of enzymatic reactions within such nanostructures, as well as staples incorporation and stability for aiding fundamental studies of enzymatic reactions in DNA nanostructures. Specifically, the restriction quantification method proposed is based on a linear PCR (L-PCR) amplification reaction that involves staple-specific carriers (70-nucleotide-long ssDNA) that can fully hybridize to staple fragments produced by the enzymatic cutting. The polymerase action leads to the formation of a duplex DNA fragment 70 base pairs (bp) long for each cleaved staple, whereas the hybridization of un-cleaved staples on the carrier prevents such polymerase reaction. To study staples incorporation efficiency, the same protocol can be used, but DNA carriers are designed to hybridize the full length of the DNA staple sequence. I prepared L-PCR samples to evaluate next-generation sequencing (NGS) quantification accuracy and L-PCR efficiency. As a proof-of-concept, I analyzed 5 staples of a triangular DNA nanostructure and obtained information on their incorporation efficiency or cleavage. This thesis also describes the design and development of a DNA based nanosensor for improving the accuracy of short nucleic acid quantification with qPCR. The work aims at coupling qPCR with a self-assembled nanosensor, which can help overcome amplification and retro-transcription reaction bias, and circumvent the detection threshold of 2-fold concentration variation, without requiring updates to traditional qPCR instrumentation. Components of such sensor are three consecutive “foot-loop” DNA probes each carrying a target-complementary sequence in the loop. Probes are assembled over a common scaffold that joins their “feet”. Each “foot” carries a restriction site and upon hybridization of three copies of the same target molecule on the respective loops, the site of each foot is destabilized (termed “bingo” configuration). Only in this case, the whole scaffold is protected from enzymatic cleavage and can be amplified with PCR. Target and bingo-scaffold concentrations are correlated by power function of 3. The results obtained demonstrate the increased accuracy of the Bingo-qPCR assay with respect to standard qPCR in evaluating small variation of enzymatic activity, and prove the feasibility of the target detection switch-based reaction.

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Choi, Kwan-yue. "A molecular epidemiology study on conjunctivitis using conventional nucleic acid amplification technologies and resequencing microarray." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B44248465.

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Choi, Kwan-yue, and 蔡君如. "A molecular epidemiology study on conjunctivitis using conventional nucleic acid amplification technologies and resequencing microarray." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B44248465.

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Bowers, Katherine. "Development and clinical performance of nucleic acid amplification techniques for the diagnosis of Strongyloides stercoralis." Thesis, University of Westminster, 2017. https://westminsterresearch.westminster.ac.uk/item/q56v6/development-and-clinical-performance-of-nucleic-acid-amplification-techniques-for-the-diagnosis-of-strongyloides-stercoralis.

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The laboratory diagnosis of Strongyloides stercoralis (S. stercoralis) at the Department of Clinical Parasitology (DCP) by the routine methods of microscopy and Strongyloides culture is not sensitive due to the, usually, low parasite load and intermittent larval excretion of the parasite. Serology (enzyme-linked immunosorbent assay) suffers from a lack of specificity because Strongyloides antibodies are known to cross- react with schistosomal, filarial and other helminthic antibodies in serological tests. Moreover, antibody levels are slow to decline after successful treatment therefore serology cannot be used to monitor point of cure. A missed diagnosis of strongyloidiasis in immunocompromised patients or those about to undergo iatrogenic immune suppression may have severe, even fatal, consequences. The disease is poorly studied because of the lack of sensitive, specific and cost-effective tests. Therefore, the decision was made to evaluate and validate nucleic acid amplification techniques (NAATs) for the diagnosis of S. stercoralis for use in a well- resourced specialist referral parasitology laboratory. A novel loop mediated isothermal amplification (LAMP) assay was also developed for use in resource- limited regions. The study was conducted over two years (2014-2016) and examined 284 residual diagnostic samples. The cohort was drawn from patients attending a central London western travel medicine (WTM) clinic. The NAATs chosen for this study were a published real- time PCR (qPCR) assay (ten Hove et al., 2009) and a novel LAMP assay. The NAATs were compared to the combined reference standard of microscopy, culture and serology for the diagnosis of S. stercoralis in stool samples. The development of the novel LAMP assay for use in resource- limited areas included the investigation of methods for rapid, simple and cost- effective DNA extraction. The qPCR and LAMP assays detect target DNA within areas on either side of the S. stercoralis 18S rRNA genome hypervariable region (Hasegawa et al., 2009). In this study the LAMP and qPCR assays demonstrated a limit of detection of 10-3 and 10-4, respectively for S. stercoralis DNA detection in clinical samples. Specificity was determined for the LAMP and qPCR assays to be 100% and 94.83%, respectively and the cost per test was calculated as £4.80 and £8.21, respectively. In this study, persistence of S. stercoralis DNA in clinical samples was improved when the samples were stored at -20oC. While the LAMP assay has a shorter turnaround time and is less costly than qPCR, the superior efficiency of qPCR detection of S. stercoralis DNA in clinical samples established that the qPCR assay was a more suitable addition to the diagnostic repertoire at a high- throughput WTM clinic. The LAMP assay showed promise for deployment in resource- limited areas and as a point- of- care test but further work is required to optimise the LAMP assay for these purposes.

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Henriksson, Sara. "Application of Padlock Probe Based Nucleic Acid Analysis In Situ." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-128446.

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The great variation displayed by nucleic acid molecules in human cells, and the continuous discovery of their impact on life, consequently require continuous refinements of molecular analysis techniques. Padlock probes and rolling circle amplification offer single nucleotide discrimination in situ, a high signal-to-noise ratio and localized detection within cells and tissues. In this thesis, in situ detection of nucleic acids with padlock probes and rolling circle amplification was applied for detection of DNA in the single cell gel electrophoresis assay to detect nuclear and mitochondrial DNA. This assay is used to measure DNA damage and repair. The behaviour of mitochondrial DNA in the single cell gel electrophoresis assay has earlier been controversial, but it was shown herein that mitochondrial DNA diffuses away early in the assay. In contrast, Alu repeats remain associated with the nuclear matrix throughout the procedure. A new twelve gel approach was also developed with increased throughput of the single cell gel electrophoresis assay. DNA repair of three genes OGG1, XPD and HPRT and of Alu repeats after H2O2 induced damage was further monitored. All three genes and Alu repeats were repaired faster than total DNA. Finally, padlock probes and rolling circle amplification were applied for detection of the single stranded RNA virus Crimean Congo hemorrhagic fever virus. The virus was detected by first reverse transcribing RNA into cDNA.. The virus RNA together with its complementary RNA and the nucleocapsid protein were detected in cultured cells. The work presented here enables studies of gene specific damage and repair as well as viral infections in situ. Detection by ligation offers high specificity and makes it possible to discriminate even between closely related molecules. Therefore, these techniques will be useful for a wide range of applications within research and diagnostics.

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13

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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Dann, Louise Claire. "Nucleic acid sequence-based amplification : relative performance and applications in HIV-1 disease monitoring and patient management." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272347.

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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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Garneret, Pierre. "Microfluidique papier pour le diagnostic de terrain : préparation d'échantillon et multiplexage." Thesis, Paris Sciences et Lettres (ComUE), 2019. https://pastel.archives-ouvertes.fr/tel-03174261.

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Depuis l’épisode EBOLA de 2014, le laboratoire MMN et de l’Institut Pasteur collaborent pour réaliser des tests de biologie moléculaire sur papier microfluidique pour le diagnostic de maladies infectieuses. Des tests réalisés à Macenta (Guinée) sur des échantillons d’ARN purifiés de patients ont démontré la pertinence de la technologie. Aujourd’hui, les deux équipes travaillent au développement d’un dispositif multiplexé (détection simultanée de plusieurs cibles biologiques). L’émergence du Virus Zika (ZIKV) transmis majoritairement par les moustiques diurnes du genre Aedes, dans des zones géographiques ou d’autres arboviroses comme la Dengue (DENV) ou le Chikungunya (CHIKV) étaient déjà présentes a récemment orienté les efforts de la collaboration vers le développement d’un test multiplexé Zika/Dengue/Chikungunya. Le but premier étant de pouvoir disposer d’un système de diagnostic de terrain rapide, peu coûteux et facile d’utilisation pour améliorer la prise en charge des patients. Le développement d’un tel dispositif serait alors mis à profit dans le réseau des instituts Pasteur pour établir un suivi épidémiologique différentiel de ces trois maladies dont la symptomatologie est identique ainsi que d’étudier la physiologie de leur co-infection
Since the Ebola outbreak of 2014, the MMN Laboratory and the Pasteur Institute are working to conduct molecular biology tests on paper microfluidics for the diagnosis of infectious diseases. Tests made in Guinée in 2015, by the two teams, have shown the pertinence of the technology. Today, both teams are working on developing a multiplexed devices (simultaneous detection of multiple biological targets). The recent Zika Virus (ZIKV) outbreak transmitted by Aedes mosquitoes in geographic area where other arboviruses such as Dengue (DENV) and Chikungunya (CHIKV) were already spreading focused the development work on a Zika/Dengue/Chikungunya multiplexed test. The first goal is to get a point of care device, cheap and easy to use allowing a better management of the patients. The development of such a device would then be used by Pasteur Institut network to monitor more precisely the epidemiology of those diseases displaying the same the same symptomatology, and study the physiology of their co-infection

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Masetty, Manaswini. "A Smartphone Enabled Molecular Diagnostic Toolkit to Detect Pathogens via Isothermal Nucleic Acid Amplification on Pre-Dried Disposable Paper Strips." University of Cincinnati / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1627664394713446.

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Hofmann, Jakob [Verfasser]. "Evaluation der klinischen Praktikabilität und prognostischen Aussagefähigkeit der intraoperativen Sentinellymphknotendiagnostik mittels One-step Nucleic Acid Amplification (OSNA) bei invasivem Mammakarzinom / Jakob Hofmann." Kiel : Universitätsbibliothek Kiel, 2014. http://d-nb.info/1053326211/34.

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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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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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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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Hadersdorfer, Johannes [Verfasser], Dieter Richard [Akademischer Betreuer] Treutter, and Thilo [Akademischer Betreuer] Fischer. "Development of an isothermal nucleic acid amplification protocol for high-throughput monitoring of Plum pox virus infection in stone fruit production / Johannes Hadersdorfer. Gutachter: Dieter Richard Treutter ; Thilo Fischer. Betreuer: Dieter Richard Treutter." München : Universitätsbibliothek der TU München, 2013. http://d-nb.info/1032313498/34.

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23

Gomez, Deborah Beltrami. "Prevalência de Chlamydia trachomatis em mulheres inférteis e gestantes assintomáticas." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2016. http://hdl.handle.net/10183/143385.

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Introdução: A infecção urogenital por Chlamydia trachomatis é a doença sexualmente transmissível bacteriana mais prevalente no mundo e afeta principalmente mulheres jovens sexualmente ativas. Infecções não tratadas podem provocar complicações reprodutivas decorrentes do dano tubáreo. Na gestação, aumenta o risco de parto prematuro, baixo peso ao nascer, morte perinatal, conjuntivite e pneumonia neonatal. Existem poucos dados brasileiros referentes à epidemiologia dessa infecção no nosso meio. O objetivo desse estudo foi determinar a prevalência de C. trachomatis em mulheres inférteis e em gestantes. Método: Foram analisadas transversalmente 77 mulheres inférteis e 60 gestantes assintomáticas. Foram coletadas amostras urinárias para ensaio de PCR e amostras sanguíneas para pesquisa de anticorpos IgG através da técnica de imunofluorescência indireta. Todas as participantes responderam um questionário referente ao seu histórico clínico e ginecológico. Resultados: A prevalência, tanto no ensaio de PCR quanto na imunofluorescência indireta (IgG) para C. trachomatis foi similar entre os grupos. Encontramos anticorpos IgG presentes em 61% das mulheres inférteis e em 56,7% das gestantes. Houve somente 1 PCR positivo no grupo das inférteis (1,3%) e nenhum do grupo das gestantes. Conclusão: Encontramos alta prevalência de anticorpos IgG para C. trachomatis em mulheres inférteis e em gestantes, mas verificamos baixa prevalência de PCR positivo nas participantes. A presença de IgG correlacionou-se com comportamento sexual e tabagismo.
Background: Chlamydia trachomatis (CT) is the most prevalent sexually transmitted bacterial infection and affects mainly young, sexually active, women. Untreated infection may lead to reproductive complications due to tubal damage. Infections during pregnancy may cause preterm labor, low birth weight, perinatal death and neonatal conjunctivitis and pneumonia. There is little data on CT infection in Brazil. The aim of this study was to determine CT prevalence on infertile and pregnant women. Methods: A cross-sectional study included 77 infertile and 60 asymptomatic pregnant women. First void urine was tested to CT using PCR and blood samples were collected for CT IgG antibodies testing using Indirect Immunofluorescence. A questionnaire about medical, gynecological and sexual history was applied to all participants. Results: We found statistically similar prevalence of PCR and IgG antibodies between groups. This study observed a 61% prevalence of CT IgG antibodies in infertile women and 56,7% in pregnant women. PCR was positive in only one (1,3%) infertile woman and in none of the pregnant. Conclusion: A high prevalence of C. trachomatis IgG antibody in Brazilian pregnant and infertile women, but a low prevalence of positive PCR on urine samples were demonstrated. CT antibodies were associated with sexual behavior and smoking.

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24

Pugnière, Pascal. "Contribution à l'amélioration de la quantification des acides nucléiques par qPCR et RT-qPCR." Phd thesis, Université de Grenoble, 2012. http://tel.archives-ouvertes.fr/tel-00870512.

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La qPCR est actuellement la technique de référence en matière de quantification d'ADN. Elle peut être définie comme une amplification exponentielle, cyclique et ciblée de la séquence d'ADN cible. Le caractère exponentiel de la qPCR est à la fois à l'origine de la sensibilité de la méthode mais aussi d'une potentielle variabilité inter-échantillons. Cette variabilité est compensée par le caractère cyclique de la méthode qui entraine une synchronisation de la réaction pour tous les échantillons à chaque cycle. L'amplification ciblée de la séquence choisie traduit quand à elle la spécificité de la méthode. Néanmoins, cette dernière propriété de la PCR reste la moins vérifiée. La spécificité de la qPCR est indiscutable lorsque la cible à détecter se trouve en quantité suffisante (>100 copies environ). En revanche, pour des quantités plus faibles ou en présence d'inhibiteurs, la spécificité diminue ou disparaît (limites de détection et de quantification). Cette perte de spécificité retentit de façon significative sur la précision et la reproductibilité du dosage à réaliser. Cependant, si l'hybridation non spécifique est inéluctable dans ces conditions, l'amplification non spécifique consécutive peut être limitée, voire supprimée au moyen d'amorces de PCR soit plus spécifiques, soit moins susceptibles de générer des différences inter-échantillons. La RT-qPCR, grâce à une étape initiale de transcription inverse (conversion d'un ARN en un ADN complémentaire) permet la quantification des ARN. Cependant, la transcription inverse reste moins reproductible que la PCR, générant des différences inter-échantillons délétères en diagnostic comme en recherche. Au cours de ces travaux, je propose des méthodes originales améliorant de façon significative différentes étapes de ces techniques. Premièrement, je propose une amélioration de la standardisation de l'étape de transcription inverse capable de diminuer de façon significative la variabilité inter-échantillons ; l'utilisation d'un volume constant d'extrait d'ARN pour chaque échantillon améliore considérablement la précision de la quantification d'ARN messagers. Deuxièmement, je propose une modification des amorces par incorporation de résidus d'acides nucléiques bloqués (LNA) ; cette modification permet d'augmenter la spécificité des amorces aux limites de la détection. Enfin, je propose une méthode simple et économique permettant la mesure directe de la température de fusion (Tm) dans les conditions réelles de la PCR. Ce paramètre, considéré comme capital pour la réalisation des dosages par qPCR, est généralement obtenu par des méthodes prédictives peu précises. De plus, cette méthode doit permettre de déterminer précisément les paramètres thermodynamiques des amorces (∆G, ∆H et ∆S) et ainsi avoir accès d'une part au pourcentage réel d'amorces hybridées en fonction de la température et d'autre part d'identifier la susceptibilité des amorces aux inhibiteurs.

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25

Maree, Hans Jacob. "Identification and characterisation of grapevine leafroll-associated virus 3 genomic and subgenomic RNAs." Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/5417.

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Thesis (PhD (Genetics))--University of Stellenbosch, 2010.
Includes bibliography.
Title page: Dept. of Genetics, Faculty of Science
ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus Ampelovirus, family Closteroviridae. There has been only one report that claimed the complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended UTR was found in all other South African isolates of GLRaV-3 that were tested. In two collaborative studies the existence of the extended 5’ UTR was confirmed and further investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended 5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the different genetic variants, however within a variant the 5’ UTR was found to be highly conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’ half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3 mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation of putative sg-promoters is also described.
AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ‘n lid van die Closteroviridae familie en die hooflid vir die genus Ampelovirus. Tot dusver was daar net een studie wat die volledige nukleïensuurvolgorde van GLRaV-3 gerapporteer het (isolaat NY-1, AF037268). In hierdie studie rapporteer ons die volledige volgorde van ‘n Suid-Afrikaanse GLRaV-3, isolaat nl. GP18 (EU259806) wat noemenswaardig langer is aan die 5’ kant. RLM-RACE is gebruik om die 5’ eindpunt van GP18 te bepaal en daar is gevind dat die 5’ ongetransleerde streek (UTR) 737 nt lank is in vergelyking met die 158 nt van die NY-1 volgorde. Die verlengde 5’ UTR is gevind in alle Suid-Afrikaanse monsters wat getoets is. Die verlengde 5’ UTR is bevestig en verder bestudeer tydens twee samewerkingsprojekte. In die eerste studie (Coetzee et al., 2010), is metagenomiese data gegenereer deur volgende-generasie volgordebepaling (Illumina Genome Analyzer II) en geanaliseer vir GLRaV-3 spesifieke volgordes. Volgordes soortgelyk aan die GP18 isolaat het die verlengde 5’ UTR volgorde bevestig. In die tweede studie (Jooste et al., 2010), is drie genetiese variante van GLRaV-3 geidentifiseer en hulle onderskeie 5’ UTR volgordes bepaal en bestudeer. Daar is groot diversiteit tussen die 5’ UTRs van die verskillende genetiese variante gevind, maar tussen isolate van dieselfde variant is die volgordes gekonserveerd. Grapevine leafroll-associated virus 3 is ‘n positiewe-sin, enkelstring RNA virus wat al voorheen bewys is om, soos ander closterovirusse, subgenomiese (sg) RNAs te produseer tydens replisering. Hierdie sgRNAs word ingespan vir die uitdrukking van die ORFs op die 3’ helfte van die virusgenoom. In hierdie studie is ‘n dsRNA klad gebruik om die voorkoms van 3’ ko-terminale sgRNAs [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] te bevestig in GLRaV-3 geinfekteerde plantmateriaal deur gebruik te maak van ‘n peiler teen die kapsiedproteïengeen. Die spesifieke 5’ terminale nukleotiedes vir hierdie sgRNAs sowel as vier additionele sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] is bepaal deur gebruik te maak van RLM-RACE op die GLRaV-3 isolaat GP18. Die konstruksie van ‘n GLRaV-3 mini-repliserings konstruk, analoog aan die RNA1 van Lettuce infectious yellows virus, vir die evaluasie van moontlike sg-promotors word ook beskryf.

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26

Gualberto, Felipe Augusto Souza. "Valor disgnóstico da nested PCR em tempo real em pacientes com meningite tuberculosa." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5134/tde-26082014-093325/.

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Introdução: A meningite tuberculosa (MTB) é a forma mais grave e fatal de tuberculose. O diagnóstico oportuno e o tratamento adequado e precoce são os principais fatores associados com o bom prognóstico. Os métodos utilizados na prática médica diária - achados clínicos, exames de imagem e análise de líquido cefalorraquidiano (LCR) - têm baixa acurácia. A pesquisa do DNA do Mycobacterium tuberculosis no LCR através da reação em cadeia da polimerase (PCR, do inglês polimerase chain reaction) com a metodologia nested é promissora, especialmente quando associada à praticidade da amplificação do DNA em tempo real. Objetivo: Avaliar o valor diagnóstico da nested PCR em tempo real (nRT-PCR, do inglês nested real-time PCR) na investigação de pacientes com MTB. Métodos: Estudo observacional realizado em duas fases: uma prospectiva e outra retrospectiva. Na fase prospectiva, foram incluídos pacientes com suspeita de MTB internados no Instituto de Infectologia Emílio Ribas (IIER). Informações clínicas, laboratoriais e radiológicas foram coletadas, assim como amostra de LCR de todos os pacientes. A partir de critérios internacionais padronizados, os pacientes foram categorizados como \"MTB Definitiva\", \"MTB Provável\", \"MTB Possível\" e \"Não MTB\". A nRT-PCR, utilizando o gene alvo mpt64, foi realizada em todas as amostras de LCR no Laboratório de Meningites Bacterianas do Instituto Adolfo Lutz. Sensibilidade, especificidade e intervalos de confiança (IC 95%) da nRT-PCR foram calculados com base no padrão-ouro (cultura positiva para M. tuberculosis ou isolamento de BAAR no sistema nervoso central) e nos pacientes com outros diagnósticos estabelecidos (Não MTB). Também foi calculada a proporção de pacientes com a nRT-PCR positiva em cada categoria clínica. Na fase retrospectiva, foi realizada uma revisão de prontuários de pacientes que tiveram a nRT-PCR solicitada no IIER e no Centro de Referência e Treinamento em DST/AIDS. Os mesmos procedimentos de categorização diagnóstica, cálculos de sensibilidade e especificidade foram adotados. Resultados: Na fase prospectiva, foram incluídos 102 pacientes, sendo 92 deles infectados por HIV. Nove deles tiveram o padrão-ouro positivo e foram classificados como \"MTB Definitiva\" e 81 deles tiveram outros diagnósticos estabelecidos (\"Não MTB\"). A sensibilidade e a especificidade da nRT-PCR foi 100% (IC95%:70-100 e 95-100, respectivamente). A positividade da nRT-PCR na categoria \"MTB Provável\" foi 50% (4/8 pacientes) e 25% na \"MTB Possível\" (1/4). Na fase retrospectiva, 56 pacientes foram incluídos, sendo 48 infectados por HIV. A nRT-PCR teve sensibilidade de 83% (5/6) e especificidade de 100% (0/45). A positividade na categoria \"MTB Provável\" foi 60% (3/5) e não houve pacientes classificados como \"MTB Possível\". Conclusão: A nRT-PCR apresentou boa sensibilidade e ótima especificidade, demonstrando seu valor diagnóstico na identificação oportuna de casos de MTB
Background: Tuberculous meningitis (TBM) is the most serious and lethal presentation of tuberculosis. Timely diagnosis and appropriated treatment are the main factors associated with good outcome. Methods used in the daily medical practice - clinical, radiological and cerebrospinal fluid (CSF) findings - have low accuracy. Search for Mycobacterium tuberculosis DNA in the CSF by polymerase chain reaction (PCR) using the nested methodology is promising, especially when combined with the practical approach of the real time DNA amplification. Objective: To evaluate the diagnostic value of a nested real-time PCR (nRT-PCR) in the investigation of patients with TBM. Methods: A two-phase observational study was carried out: prospective and retrospective. In the prospective phase, patients with suspected TBM hospitalized at \"Instituto de Infectologia Emílio Ribas\" (IIER) were included. Clinical, laboratory and radiological data were collected, as well as CSF samples of all patients. According to international standard criteria, patients were categorized as \"TBM Definite\", \"TBM Probable\", \"TBM Possible\" and \"Not TBM\". The nRT-PCR, using the mpt64 gene, was performed on all CSF sample in the Laboratory of Bacterial Meningitis, Adolfo Lutz Institute. Sensitivity, specificity and confidence intervals (95% CI) of the nRT-PCR were calculated based on the gold standard (culture positive for M. tuberculosis or AFB isolation on the central nervous system) and on patients with other established diagnoses (\"Not TBM\"). The proportion of patients with a positive nRT-PCR in each clinical category was also calculated. In the retrospective phase, medical chart review was performed in those patients who had the nRT-PCR requested in IIER and in the \"Centro de Referência e Treinamento em DST/AIDS\". The same diagnostic categorization and calculations of sensitivity and specificity were adopted. Results: 102 patients were included in the prospective phase, 92 of them HIV-infected. Nine of them had the gold standard positive and were classified as \"TBM Definite\" and 81 of them had other diagnoses established (\"Not TBM\"). The sensitivity and specificity of the nRT-PCR were 100% (95%CI: 70-100 and 95-100, respectively). The nRT-PCR positivity in category \"TBM Probable\" was 50% (4/8 patients) and 25% in \"TBM Possible\" (1/4). In retrospective phase, the nRT-PCR had a sensitivity of 83% (5/6) and specificity of 100% (0/45), among the 56 included patients (48 of them HIV infected). Positivity in \"TBM Probable\" category was 60% (3/5) and no patients were classified as \"TBM Possible\". Conclusion: The nRT-PCR showed good sensitivity and excellent specificity, showing its diagnostic value in the timely identification of TBM

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27

Priyanka, V. "Droplet Isothermal Amplification For Nucleic Acid Quantification." Thesis, 2022. https://etd.iisc.ac.in/handle/2005/5643.

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Nucleic acid quantification (NAQ) is extensively employed for gene expression analysis, monitoring viral loads, detecting rare or dysfunctional cells, and assessing treatment regimes. The gold standard, quantitative polymerase chain reaction (qPCR), and the recent alternative, droplet digital PCR (ddPCR), provide accurate quantification of nucleic acids (NA). Albeit the requirement of thermal cycling and separate platforms for droplet generation, NA amplification, and signal detection, in the case of ddPCR increases the assay complexity and time, limiting its broad applicability. In this work, we have developed an integrated droplet isothermal amplification-based NAQ (idNAQ) platform that enables facile and fast NAQ with a large dynamic range. First, we adapted the isothermal amplification method, Recombinase Polymerase Amplification (RPA), for NAQ. We demonstrate a fast (• 40 minutes) semi-quantitative RPA (qRPA) assay with the endpoint intensity ratio (EIR) for DNA quantification with a 6-log order range. Since the EIR model estimates the amplicon levels at the end of the reaction, real-time monitoring of the amplification reaction (unlike in the case of qPCR) is no longer required. With qRPA, we demonstrate viral load detection from the serum of dengue-infected patients with comparable performance to qPCR. The later section discusses the translation of the qRPA NAQ to a microfluidic droplet format. Droplet RPA (dRPA) displays similar kinetics to the bulk reaction suggesting successful optimization of droplet conditions for RPA. dRPA in the low concentration regime follows Poisson distribution that enables digital quantification as in the case of ddPCR. On the other hand, at a higher starting concentration of DNA (non-digital regime, >10 DNA per droplet), the RPA amplification in droplets exhibits heterogeneous intensity puncta due to rapid amplification and incomplete mixing leading to the formation of DNA ‘amplification globules’. We use a supervised machine, learning-based regression model with these intensity features as inputs to accurately predict the target concentration of up to 10^5 molecules per droplet. Combining these two modalities of dRPA yields a dynamic range of >7 log orders of concentration that are comparable to qPCR. Finally, we demonstrate the successful integration of all unit operations onto a single microfluidic device for droplet RPA and quantification. Different microfluidic designs were optimized for monodisperse droplet generation and image acquisition from a large incubation area that allowed the successful implementation of quantitative dRPA in a single device.

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28

Jue, Erik Bradley. "Improved Tools for Point-of-Care Nucleic Acid Amplification Testing." Thesis, 2020. https://thesis.library.caltech.edu/13735/1/200529_erik_jue_2020_thesis_final.pdf.

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There is a critical need for improved diagnostic tools to detect infectious diseases, especially in low-resource regions. A sample-to-answer point-of-care nucleic acid amplification test (NAAT) would be incredibly valuable for many different applications (e.g. COVID-19, Chlamydia/Gonorrhoeae, Influenza, Ebola, Zika/Chikungunya/Dengue, etc.). However, sample preparation (purification of pure nucleic acids) is a challenging bottleneck. In Chapter 2, commercial NA extraction methods were studied and improved. In Chapter 3, commercial stocks of SARS-CoV-2 RNA used in FDA emergency-use authorizations were found to be inaccurate and were independently quantified using reverse transcription digital PCR. In Chapter 4, a 3D printed meter-mix device was developed for initial processing prior to the sample preparation device. In Chapter 5, a 3D printed sample-to-device interface was prototyped to facilitate loading multi-volume SlipChip devices with purified template mixed with LAMP reactants. In Chapters 6-7, advancements were made for image processing of commercial chips to study digital LAMP reactions. In Chapter 8, additional tools were developed towards sample-to-answer point-of-care NAAT including a sample preparation module, amplification module, cell-phone readout, and automated base station.

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29

Hsieh, Tsung-Min, and 謝宗閔. "Micro Thermocyclers for Nucleic Acid Amplification with High Thermal Uniformity." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/30145900653046174870.

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博士
國立成功大學
電機工程學系碩博士班
96
The trend of miniaturization of biomedical instruments began in the last century, and is crucial for furthering progress in medical technology. In this study, an integrated chip–PCR/RT-PCR system is implemented successfully, meeting the requirements for use in miniature thermocyclers – small in size, low power consumption, high heating and cooling rates, and even portability, with resistance temperature detectors and thin-film micro-heaters. Furthermore, due to the modifications made to the micro thermocycler, the thermal uniformity of a specific region has been greatly enhanced, improving the performance of the micro device. The initial approach of this study was to develop a block-type thin-film micro-heater and resistance temperature detectors on a glass substrate to form a micro thermocycler. Based on the techniques of feedback control and pulse width modulation, PCR thermal cycling was achieved, and the system was tested using Salmonella, with successful results. Two new approaches were then implemented and applied to increase DNA amplification efficiency and reduce non-specific PCR products. In order to achieve these aims, first, a novel kind of micro thin-film heater, an array-type micro-heater, was designed and developed. The main advantages of array-type micro-heaters are their lower resistance as compared with block-type heaters and their provision of greater thermal uniformity with a uniform heating density. Active compensating units were also added and applied in order to reduce the edge-effect, i.e., thermal loss from the edges. From these array-type micro-heaters and active compensating units, a miniature thermocycler with enhanced thermal uniformity was developed and the success of its performance verified with S. pneumoniae. The second approach was the design of a novel self-compensating array-type micro-heater. This kind of micro thin-film heater not only increases the thermal uniformity in the specific heating area, but also eliminates additional control themes on thermal compensating. Based on self-compensating array-type micro-heaters, this device is of a fully two-dimensional thermal compensating design used in thin-film heaters. Experimental results from infrared images showed that the percentage of the uniformity area with a thermal variation of less than 1�aC was 90.3%, 99.9% and 96.8% at PCR thermocycling temperatures of 94�aC, 55�aC and 72�aC, respectively, which represents a significant improvement over conventional block-type or serpentine-shaped micro-heaters. The performance of the chip–PCR system based on self-compensating array-type micro-heaters was assessed by amplifying a detection gene (171bp) associated with the dengue virus serotype 2, the results of which were successful, the amplification efficiency having been found to be statistically greater than that of other micro-heaters without a self-compensation function. The micro-heater proposed in this study was based on the aim of finding an efficient approach to greatly improve the thermal uniformity of a specific micro area requiring precise thermal conditions, and a micro thermocycler for nucleic acid amplification with high thermal uniformity was developed. This system not only satisfies the requirements of micro biomedical devices, but could be also useful in the design of a micro-total-analysis-system and a lab-on-a-chip, which require high thermal uniformity in order to improve the performance of the miniature devices.

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30

Drake, Philip, Y.-C. Chen, I. Lehmann, and P.-S. Jiang. "Nanoparticle labels for pathogen detection through nucleic acid amplification tests." 2014. http://hdl.handle.net/10454/10333.

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Yes
Magnetic nanoparticles and surface-enhanced Raman scattering (SERS) active nanoparticles were coated with short chain DNA tags. These were then used to identify a target bacterial DNA sequence. The tags function as primers in a standard PCR with the reverse primers and forward primers on the SERS nanoparticles and magnetic nanoparticles, respectively. During the PCR cycles, a composite nanostructure is formed that is both magnetically responsive and SERS active. After magnetic trapping, the intensity of the SERS signal can be related back to the concentration of the target DNA. A test assay was performed that showed a detection limit (based on the signal to noise ratio) of less than 3 zeptomole (41 pg/L). For comparison, a PCR assay based on the standard SYBR Green method was performed. This used the same primers and target DNA and had a detection limit of 10 attomoles (138 ng/L), 3,000 times less sensitive. The work documents the proof of principle study and shows for the first time the use of SERS-NP labels in the quantification of nucleic acid amplification tests and PCR.

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31

Ping-Hua, Teng, and 鄧秉華. "Application of isothermal nucleic acid amplification on shrimp viral disease diagnosis." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/39938024179183265321.

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博士
東海大學
畜產與生物科技學系
97
Shrimp viral diseases are important issues in aquaculture industries. Until now, there have been not efficient vaccines or therapeutic strategies against these viral diseases. To face the threats, farmers tried to decrease cultivation risks with more efficient methods. However, they had no tools to early detect and prevent viruses into cultivation environment. For the purpose of pathogen detection before disease outbreak, a diagnostic platform with efficiency and accuracy plays an important role for health management of shrimp culture. Traditional strategies, such as histochemistry or immune-related techniques, only could be used as disease determination because of their insufficient sensitivities. To pathogen prevention, however, applications of molecular diagnostic technologies are feasible strategies to conquer the defects of sensitivities and complexities of histochemistry and immuo-related techniques. Polymerase chain reaction (PCR), for example, is a powerful diagnostic tool that can detect even 10 molecules in the reaction. PCR related techniques had been used to establish the culture system of specific pathogen free (SPF) and daily diagnosis for large-scale or integrated farms. However, the needs of equipments and technicians for a PCR laboratory operating are too expensive for medium or small-sized farms. Although some diagnostic centers may provide services of PCR diagnosis, the time to get results is always too long. With so many difficults to get efficient diagnostic tools, the pathogenic threats are accomplished with these smaller farms. In the study, some isothermal amplification techniques will be applied to develop a simple, economic and accurate molecular diagnostic platform to match farmers’ needs. Isothermal amplification techniques, including ramification amplification (RAM), nucleic acid sequence based amplification (NASBA) and loop-mediated isothermal amplification (LAMP), will be evaluated the feasibilities. In addition, real-time detection of these products amplified by isothermal amplifications is also tested to avoid post-amplification procedures and simplify data interpretation. The results showed that the sensitivity of RAM in detecting IHHNV was 100 copies per reaction, and the sensitivity of NASBA and reverse transcription LAMP in detecting TSV was 50 and 100 copies per reaction, respectively. Three kinds of isothermal amplification methods could be achieved high sensitivities comparable to that of PCR at 3 hours. The specificities and simplicities of these methods are also verified under different conditions to match the purpose of on-site use. For the purposes of real-time detection and quantification, LAMP coupled with fluorescence resonance energy transfer (FRET) techniques is studied and the results are available for follow-up developments. For further utilities, a novel real-time isothermal amplification detection machine should be developed to fit the purpose of diagnosis with simple, economic and accurate.

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32

Panwar, Jatin. "Droplet Microfluidics for Nucleic Acid Quantification and Single Cell Analysis." Thesis, 2019. https://etd.iisc.ac.in/handle/2005/5018.

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Droplet microfluidics provides controlled generation of monodispersed droplets of the order of a few picoliters in multiphase microfluidic systems. These droplets are employed as micro-reactors to conduct chemical/biochemical reactions and assays in a controlled and high-throughput manner that find applications in point-of-care and lab-on-chip platforms. While the microfluidic devices are compact, the existing solutions to control fluid flow operations have a significant footprint that effects their portability, logistic viability and economics. As an alternate to the existing instrumentation-intensive flow-rate driven control for droplet generation, we studied and standardised suction driven fluid-flow control. In multiphase and multi-channel devices with suction-based flow control, microchannel geometry and suction pressure at the outlet determine the flow rates in individual channels. It is thus critical to understand the role of geometry along with suction pressure in the dynamics of droplet generation. We propose a governing parameter, called as modified capillary number, that captures droplet generation behaviour and outlines the design requirements for a suction driven droplet generation. As droplet microfluidics allows capture and analysis of individual cells with unprecedented control and throughput, single cell studies with microdroplets are gaining popularity. However, such analysis requires microfluidic devices with multiple unit operations that become a challenge with suction driven fluid-flow due to limited pressure head and lack of independent control over dispersed and continuous phase flow rates. To demonstrate single cell analysis, we defined and developed individual unit operations integrated in a multi-operation suction microfluidic device designed to quantify the low copy number RNA from single cells. The device, droplet digital Single cell Nucleic Acid Quantifier (dd-ScNAQ), successively performs encapsulation of single cells in droplets, cell lysis and cellular lysate/RNA distribution in smaller secondary droplets followed by on-chip temperature controlled isothermal nucleic acid amplification for quantification using fluorescence microscopy in a continuous flow geometry. Finally, to bypass the optical methods for detection of cells in droplets, which limits ‘in-field’ microfluidic applications; we developed a low-cost microfluidic impedance cytometry (MIC) approach for single cell quantification. We devised a rapid micro-fabrication protocol for flow devices with integrated coplanar in-contact field’s metal (icFM) electrodes to conduct MIC. With a single-step photolithography protocol, our icFM electrodes provide a cost-effective alternate to the conventional clean-room intensive microelectrode fabrication processes. The performance of icFM electrodes, averaged over a frequency range of 0.5 to 4 MHz, is found to be comparable to the widely used platinum electrodes in the detection of single human erythrocytes in a feedback-controlled suction driven MIC setup. However, the two electrodes show frequency dependent variability during impedance measurements that is attributed to the contrast between double layer capacitance and effective charge densities at their respective surfaces. We also demonstrate quantification of single cells entrapped in water-in-oil droplets using our icFM MIC devices as a non-optical and label-free method for droplet based single cell analysis. Together, this study extends the reach of existing microfluidic technologies to address the needs of the rapidly growing lab-on-chip industry by presenting novel methods to conduct microfluidic operations for single cell studies.

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33

Liao, Chia-Sheng, and 廖家陞. "Automatic Nucleic Acid Amplification Microsystems for Rapid Pathogen Diagnosis of Infectious Disease." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/69312723014061329434.

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碩士
國立成功大學
微機電系統工程研究所
93
Rapid advances in biochemistry and bio-genetics lead to molecular biology, which is revolutionizing scientific thought. A micro-electro-mechanical-system (MEMS) allows human manipulation of micrometer-scale molecules. Advancement in biotechnology will hopefully improve the quality of human life. 　This investigation presents an automatic rapid diagnosis microsystem based on nucleic acid amplification. The miniature system is fabricated using MEMS techniques, and consists of a micro temperature control module and a microfluidic control module. The heating and temperature sensing elements of micro temperature control module are both fabricated using platinum, and are located within the reaction chambers to generate rapid and uniform thermal cycling. The microfluidic control module, including the reservoir, micropumps, microvalves and microchannel, enables automatic testing with minimal human intervention. 　This study proposes a microsystem to detect several genes associated with the DNA-based upper respiratory tract infection microorganisms, their corresponding antibiotic-resistant gene and RNA-based virus. An efficiency multiplex amplification of many targets of interests in one reaction is feasible using the proposed micro device. Additionally, multiple PCR, a novel approach of processing of various samples with different thermal cycle conditions in parallel, is developed for fast diagnosis. Multiple PCR is much faster than the traditional bench-top PCR machine system. The proposed microsystem also realizes a two-step reverse transcription polymerase chain reaction. The complete detection process, including cell lysis, sample/reagents transportation, cDNA synthesis and amplification, can be automatically performed in a user-friendly manner. 　The experimental data demonstrate that the high heating/cooling rates of the microsystem (20℃/sec and 10℃/sec, respectively) permit successful amplification of DNA microorganisms within 15 minutes and of RNA virus within one hour. The portable microsystem is packaged and operated by a 9-volt battery. The developed microsystem provides a crucial tool for many applications such as genetic analysis, molecular biology and infectious disease detection.

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34

Yang, Litao. "Coupling aptamer biosensors to signal amplification." Thesis, 2007. http://hdl.handle.net/2152/3098.

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35

Yang, Litao 1976. "Coupling aptamer biosensors to signal amplification." 2007. http://hdl.handle.net/2152/13284.

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36

Chou, Wen-Pin, and 周文彬. "Development of a Novel Nucleic Acid Amplification System-Capillary Convective Polymerase Chain Reaction." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/19692785520165047927.

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Анотація:

博士
國立臺灣大學
機械工程學研究所
99
A typical polymerase chain reaction (PCR) is a process containing three temperature steps for denaturation, annealing and elongation. In a traditional thermal cycler, metal block is used to heat or cool the reaction tubes, and more than two hours are needed for 45 cycles of the repeat heating and cooling steps. However, expensive thermal controller and long time-consuming made thermal cyclers are not suitable in self-test for personal use. In this study we describe a new method for DNA amplification using a principle of convection. In this platform, only a dry bath is required for heating the bottom of the reaction tube. Then the tube is cooled by the surrounding air by which the reagent in the tube forming convection repeatly. Thus, reaction mixture in the tube should undergo the three steps of PCR in the convection. This kind of convection in the tube is just like the phenomenon during water is boiled and it makes the reagents in the tube should fluid spontaneously to achieve different temperatures. This technique can shorten the amplification time prominently and solve the traditional problem for long time-consuming process during heating and cooling. By this way, no expensive equipment is needed and it makes this new method feasible to be used in almost all the laboratories. The key point of successful working of this platform is how to make the fluid reagent mixture of PCR reaction in the tube to form a stable and efficient convection. Here we focus on two important parameters. One is the ratio between height and diameter of the reaction tube, which determines whether the temperature gradient from the bottom to top can fulfill the three-step temperatures needed in the PCR reaction. The other is the criterions of the reagents designed in convective PCR. Because reagents in convective PCR reaction undergo a continued temperature gradient which is different from traditional PCR, it requires particular criterions for reagents in order to improve the level of yield and the rate of success. In this study, we also provide the physical parameters and design criterions of particular reagents for successful convective PCR. Using this newly established method, DNA amplification could be completed within 25 minutes and the sensitivity was measured to reach 30 copies/per tube. Besides, specific reagents were also designed for various virues, including new influenza virus H1N1, white spot syndrome virus (WSSV), and polyomavirus (BKV). Moreover, we also explore whether primers conjugated with LNA should avoid side products in the amplification. We will focus on developing a cheaper and more convenient technique for examination using our established platform on site, where it could be operated by persons without well-training. We also believe this study will provide a new direction and will be helpful and useful in the future in controlling infectious disease for human, animal and plants.

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37

Loan, Thomas. "Cell Lysate as a Platform Technology for Biocatalytic Synthesis and Nucleic Acid Amplification." Phd thesis, 2020. http://hdl.handle.net/1885/204831.

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The research presented in this Thesis focuses on the enzymatic synthesis of value-added products using cell lysates prepared from E. coli as low-cost biocatalysts. Naturally abundant endogenous enzymes are leveraged where possible and these are augmented with heterologous proteins, incorporated by recombinant overexpression, to create multi-enzymatic cascade reactions. A robust ATP regeneration system was developed, utilizing the endogenous acetate kinase (Ack) with acetyl phosphate. Application to biocatalytic synthesis of UTP from uridine was demonstrated by incorporation of a recombinant uridine kinase (Udk). This cell lysate catalyzed the one-pot synthesis of UTP, CTP and 5-fluoro-UTP from the respective nucleosides, with only substoichiometric amounts of ATP when acetyl phosphate was added to the reactions. These endogenous enzymes were also applicable to the synthesis of all naturally occurring dNTPs and NTPs from the corresponding monophosphates, and these could be employed in biotechnological processes that require the triphosphates for synthesis of nucleic acids. This Ack-mediated ATP regeneration strategy was then applied to the synthesis of dNTPs from stable, unphosphorylated deoxynucleosides, utilizing a recombinant deoxynucleoside kinase (dNK). This was optimized for direct combination with PCR, enabling a one-pot dNTP synthesis to PCR process. Moreover, by co-expression with a DNA polymerase, a cell lysate was prepared from a single culture that contained all the necessary enzyme activities for dNTP synthesis and PCR. Cell lysate was also utilized for the one-pot synthesis of UTP from low-cost biological building blocks, specifically ribose and nucleobases. This led to the development of an efficient cell-lysate-based multi-enzymatic cascade reaction for the synthesis of UTP via phosphoribosyl pyrophosphate (PRPP). This cascade was high yielding, producing 1.4 g.L 1 UTP from equimolar amounts of ribose and orotic acid. This biosynthetic strategy was also applicable to the synthesis of ATP and GTP from adenine and guanosine respectively. Finally, the application of lysate-mediated ATP regeneration to carboxylation was investigated, by recombinantly overexpressing acetyl CoA carboxylase (ACC). As part of this investigation endogenous phosphotransacetylase (Pta) in cell lysate was employed for biocatalytic synthesis of acetyl CoA from acetyl phosphate. This led to the development of a cell-lysate-mediated synthesis of 3-hydroxypropionate from acetyl phosphate, utilizing recombinant ACC and malonyl CoA reductase (MCR) in combination with the endogenous Pta. Attempts to improve the productivity of this cascade by incorporating a heterologous ACC led to the development of a strategy for post-translational biotin addition to heterologous ACC subunits in E. coli. Overall, this work serves to demonstrate the potential of cell lysates as a platform for synthetic biology and expands the scope of their application.

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38

DAI, NING, and 戴寧. "Development of Thermostatic Nucleic Acid Amplification Technology for Detecting Acute Hepatopancreatic Necrosis Disease (AHPND)." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/06651700425244830036.

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碩士
國立高雄海洋科技大學
海洋生物技術研究所
104
The outbreak of acute hepatopancreatic necrosis disease (AHPND) from 2009 has caused mass losses in shrimp farms worldwide. The strain of Vibrio parahaemolyticus which obtain a plasmid and secret toxins has been determined as the infectious agent. PCR methods were published and used to perform diagnosis in laboratory. This study developed methods of Loop-mediated isothermal amplification (LAMP) and Recombinase polymerase amplification (RPA) to detect AHPND and aimed to carry out the diagnosis at the farm side. Compared to PCR, the nucleic acid amplification of LAMP and RPA is performed at a constant temperature which shortens the reaction time and excludes complex equipments. The results are directly color presented by fluorescent dye or lateral flow dipstick which substitute for the gel electrophoresis. The LAMP system for detection of AHPND have six primers targeting toxin A gene (333 bp) in the plasmid under 65 ℃condition. LAMP results were observed through the stain of fluorescent dye and showed the color change emitted by ultraviolet light. RPA have designed two methods for amplification of the target sequence, one is using two labeled primers, the other is added the probe to increase the specificity. After reaction at 37 ℃, both methods followed with lateral flow dipstick which antibody labeled recognize RPA amplicons. Combined with the DNA easy extraction method for template preparation, the time needed to complete the diagnosis is only 1-2 hours. The sensitivity and specificity of LAMP and RPA are better or comparable to PCR in this study. We also examined several white shrimps (Litopenaeus vannamei) from Kaohsiung and found positive signals. It revealed the distribution and consideration of AHPND.

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39

Lien, Kang-Yi, and 連剛逸. "Miniature Microfluidic System Integrated with a Sample Pretreatment Device for Rapid Nucleic Acid Amplification." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/84844620753199418940.

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碩士
國立成功大學
奈米科技暨微系統工程研究所
95
Recently, purification and enrichment of bio-samples is crucial for the analysis of biosamples with an ultra-low concentration that the performance of the detection system can be efficiently increased. Apart from that, clinical samples usually contain biological medium that would normally inhibit the subsequent ribonucleic acid (RNA)/deoxyribonucleic acid (DNA) amplification process. Moreover, there still need several tedious purification and washing steps and labor-intensive processes to complete the sample preparation. In addition, a number of large-scale equipments are usually needed in the complex pretreatment procedures and the bio-samples may also be wasted and cause some contaminations during manual operations. To improve this, the concept of MEMS (micro-electro-mechanical-system)-based miniaturization is applied in the magnet beard-based sample purification and enrichment process. It is generally believed that the miniaturized bio-devices typically bring the advantages of shorter diagnosis times, less sample and reagent consumption, improved resolution, higher sensitivity, and lower cost. By the incorporation of the microfluidic system, the miniature bio-devices provide the powerful techniques to transport or mix the fluids in the system and make the biological diagnosis a quick and automatic process. Nevertheless, the integrated systems still need other detection chambers to perform biological diagnosis. And the on-chip microfluidic modules still need several sets of unidirectional microfluidic structures to complete the whole process. As a result, several electromagnetic valves (EMVs) are usually required to deliver the fluid in the microchannels and the manual operations are normally required since the lack of function of sample pretreatment in the micro system. Consequently, there is a need for a sample pretreatment process by utilizing a microfluidic system to automate the nucleic acid amplification process with less human intervention and also to improve sensitivity and selectivity. The study therefore proposes three new miniature reverse transcription polymerase chain reaction (RT-PCR) systems that integrate with bio-sample pretreatment devices using superparamagnetic beads into a single chip platform. In the first system, three modules were integrated including a microfluidic nodule consists of three sets of novel pneumatic micropumps, a bead collection module with Au wires and a micro temperature control module for the polymerase chain reaction (PCR) process. For the second system proposed, the microfluidic chip integrated two functional devices for bio-samples purification and enrichment including pumping, mixing and separation by utilizing a rotary microfluidic module and a bead-collection module consists of 2-dimension/3-dimension (2-D/3-D) microcoils. The original rotary microfluidic module can provide a rapid flow pumping rate and a high mixing efficiency and can pretreat the bio-samples in a short period of time. The third microsystem uses the novel microfluidic system including a two-way serpentine-shape (s-shape) micropump requiring only one EMV to transport and to mix the bio-samples in the microchannels. In addition, new bio-separators are developed either to perform the separation of magnetic beads or to control the temperature field for the subsequent RT-PCR process. The rapid heating/cooling rate of the micro-heating chambers can significantly shorten the pre-treatment and diagnosis processes. As a whole, the developed system may provide a powerful platform integrating the functions of sample pretreatment and fast disease diagnosis.

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40

Chen, Wei-Jang, and 陳位彰. "The Design of Temperature Control Mechanism and Its Function on Nucleic Acid Quantification." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/fj79hp.

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碩士
國立臺北科技大學
能源與冷凍空調工程系碩士班
96
This Real Time PCR machine allows the detection of DNA amplification through out the detection of the fluorescence labeling dye in the PCR mix during the early phase of this reaction. In addition, the concentration of target DNA fragment in the PCR mix before thermal cycling can be obtained from the time recorded history of the fluorescence intensity by integrating thermal cycler and fluorimeter. The Real-Time PCR machine has higher sensitivity and consumes less time than those of the traditional PCR machine. This study modified the temperature control mechanism developed by the previous studying results for the optimization of DNA amplification. A novel PID control program coded by Visual Basic was proposed to achieve optimized control of thermocycling of PCR. By 2% Agarose gel electrophoresis and UV absorption detection system, the yield products from this novel machine and the ABI 9700 PCR instrument were compared to verify the stability and reliability of the machine developed by our team. This results show that the temperature control scheme proposed in this study can be successfully applied in nucleic acid qualitative analysis. The 103 copies / ml initial copies sample has been successfully amplified. The 108 and 106 copies / ml samples were amplified successfully with high reproducibility. Regarding to the fluorescent detection system, a novel optical design was also proposed in this study. Because of the very weak fluorescent from the labeling dye attached on the DNA double strands, the fluorescent detection device itself should have very low dark current and the environmental light interferences should be avoided. The optical structure with black body painting surface was constructed to adopt cooled CCD based fluorescent detection devices to reduce the noise of fluorescent signal detection and isolate the sample under detection for high signal to noise ratio measurement of the fluorescence during PCR. The detection limit of this fluorescent detection system is 1 fmol Fluorescein. The signal can be resolved with the readings of the water. To summarize, this study has constructed a Real Time PCR machine for DNA amplification and quantification. In the future, we this instrument can be commercialized for a low cost and high accuracy DNA quantification solution.

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41

Chen, Lin [Verfasser]. "Miniaturised nucleic acid analysis systems : purification, amplification and real-time detection / vorgelegt von Lin Chen." 2007. http://d-nb.info/997471891/34.

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42

Huang, Fu-Chun, and 黃富駿. "An Integrated Microfluidic System for Nucleic Acid Amplification, Electrophoresis Separation and On-line Optical Detection." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/38805165998727799529.

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Анотація:

博士
國立成功大學
工程科學系碩博士班
96
This dissertation presents an integrated microfluidic chip capable of performing DNA/RNA (Deoxyribonucleic acid/Ribonucleic acid) amplification, electrokinetic sample injection and separation, and on-line optical detection of polymerase chain reaction (PCR) products in an automatic mode. In the device introduced here, DNA/RNA samples are first replicated using a micromachine-based PCR module or reverse transcription polymerase chain reaction (RT-PCR) module and then transported by a pneumatic micropump to a sample reservoir. The samples are subsequently driven electrokinetically into a microchannel, where they are separated electrophoretically and then detected optically using an optical fiber integrated into the device. The various modules of the integrated microfluidic chip are fabricated from cheap biocompatible materials, i.e. polydimethylsiloxane, polymethylmethacrylate and soda-lime glass. The functionality of the device is demonstrated through its successful application to the DNA-based bacterial detection of Streptococcus pneumoniae and the RNA-based detection of Dengue-2 virus. It is shown that the low thermal inertia of the PCR/RT-PCR modules reduces the sample and reagent consumption and shortens the reaction time. In order to further decrease the sample volume and to enhance disposability, a uniform temperature chip for PCR and capillary electrophoresis (CE) chip sealed with a polyethylene/thermoplastic elastomer (PE/TPE) film are developed. The uniform-temperature PCR chip was fabricated on soda-lime glass using MEMS technologies. A fence-type design of micro-heaters was used to improve the thermal uniformity of the reaction chamber. Experimental data show that the temperature distribution of the reaction chamber (diameter is 3 mm) is less than 1.0℃ in the PCR chamber at 52.0℃. The PE/TPE film packaged CE chip is performed at atmospheric pressure and room temperature, which is a fast, easy and reliable bonding method to form a sealed CE chip. The fabrication of polymethylmethacrylate (PMMA) and polycarbonate (PC) microfluidic channels is accomplished by using an injection-molding process, which could be mass-produced for commercial applications. In addition to microfluidic CE channels, three-dimensional reservoirs for storing bio-samples, and CE buffers are also formed during this injection-molding process. The functionality of the mass-produced CE chip is demonstrated through its successful separation of �珴-174/HaeⅢ DNA markers within 2 minutes. The integrated microfluidic device proposed in this study represents an important contribution to the fields of molecular biology, genetic analysis, infectious disease detection, and other biomedical applications.

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43

Wang, Tzu-Chia, and 王子嘉. "Integrated Nucleic Acid Extraction, Amplification, and Detection on Paperfluidics for Diagnostics of Orchid Virus Disease." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/sk5w8v.

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碩士
國立臺灣大學
機械工程學研究所
107
In the study, we combined the extraction, amplification, and detection of nucleic acids in molecular diagnostics, and integrated them into paper-based microfluidics (paperfluidics) which was low-cost, lightweight, and portable. Paperfluidics reduced the complicated process and operation time compared to traditional molecular diagnostic technology and did not require precise instruments. This study developed point of care (POC) device that combined known technology and innovational methods, which could diagnose two major orchid viruses. First, the results showed that elliptic paperfluidics made by cellulose-based paper could capture and concentrate nucleic acids rapidly. The extraction process took only 10 minutes which was 12 times faster than using normal column purification. The second step was to amplify targeted virus genes by using recombinase polymerase amplification (RPA) at a constant temperature of 39°C. Compared with conventional amplification, polymerase chain reaction, RPA merely takes 30 minutes to amplify. Finally, using commercially available lateral flow strips for detection, we demonstrated the ability of the device to detect the two viruses CymMV and ORSV. Meanwhile, the results of the amplification reaction were analyzed by agarose gel electrophoresis. The best buffer was adding guanidine thiocyanate to extraction. Moreover, isothermal amplification and lateral flow assay integrated on the paper-based microfluidics achieved simple and rapid molecular diagnostics. The study successfully transformed molecular diagnostics into a portable paperfluidic device. We hope that the paper-based device will be used not only in the laboratory but also in the environments with limited resources such as farmland.

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44

Huang, Hsuan-Shian, and 黃宣憲. "Nucleic acid detection, quantification and sequence analysis of canine distemper virus from clinical specimens." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/95670168567838042013.

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Анотація:

碩士
國立臺灣大學
獸醫學研究所
93
Canine distemper virus (CDV) infection in dogs can result in subclinical infection, gastrointestinal signs, and/or respiratory signs, frequently with central nervous system (CNS) involvement, high morbidity and mortality. Recently, the incidence of canine distemper (CD) both in unvaccinated and vaccinated dogs seemed increasing in Taiwan. In order to understand more about the current CDV infection in Taiwan, a rapid and sensitive diagnotic test for CD using a RT-PCR combined with nested PCR was applied to 440 dogs clinically suspected with CDV infection. 660 clinical specimens including CSF, whole blood, nasal swab, ocular swab, rectal swab and urine were collected from 440 dogs. The results showed that Nested PCR (37.7%, 166/440) increase 20.7% in the efficiency of the diagnosis than RT-PCR (17.0%, 75/489) (P<0.001). The seasonal distribution of the infection was mainly seen in late autumn to winter (P<0.001). Detection rate of conjunctival scraping (100.0%, 11/11) had a significant elevation compared with whole blood (64.4%, 38/59) (P<0.05). Quantification of the viral RNA concentration in clinical specimens from 27 cases by real-time PCR revealed that the highest concentration was as high as 1.8 x 1010 copies/mL. Different specimens from the same animal could vary up to 109 times. Nasal swab, ocular swab, oral swab and rectal swab had higher viral load, indicating that virus was shed in respiratory tract, digestive tract and urinary tract correlated to their clinical syndrome. Nasal swab was found to have the highest viral copies in cases with or without viremia. Therefore, the blood is not the best choice for clinical diagnosis. The result of H gene sequence analysis showed, that Taiwanese CDV is in the lineage of CDV Asia-1.

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45

Ho, Ya-Lun, and 何亞倫. "Development of a Surface Acoustic Wave Based Micro-Droplet Control System and its Application of Nucleic Acid Amplification." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/43732841823831743871.

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碩士
國立臺灣大學
應用力學研究所
99
In this thesis, an automatic micro-droplet control system applied to amplification of nucleic acid is accomplished by the combination of a surface acoustic wave (SAW) device, micro-heaters, micro-sensors of temperature, and a PI controller. The SAW device constituted of slanted finger interdigital transducers (SFITs) is utilized to actuate the micro-droplet by the acoustic streaming and to detect the micro-droplet by the frequency responses of the SAWs. With the development of PI controller, the micro-droplet can be manipulated automatically. In order to reduce the driving power and manipulate the mineral oil which is necessary for the reaction of the nucleic acid amplification, a perﬂuoroalkylsilane (PFAS) and tetraethoxysilane (TEOS) hydrophobic film is utilized for surface modification. Furthermore, with the PFAS/TEOS hydrophobic film, the cross-contamination can be prevented, and the micro-droplet control system is reusable for different DNAs and reagents. Utilizing the developed micro-droplet control system, the amplification of nucleic acid can be successfully achieved. The consumption of the biomedical reagent and the production cost of devices can be reduced, and the efficiency of reaction can be improved. The developed micro-droplet control system is suitable for various types of lab-on-a-chip system in biomedical fields.

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46

Chen, Jiann-Mou, and 陳建謀. "A Rapid and Real-time Nucleic Acid Detection System by Fluorescence Resonance Energy Transfer and Isothermal RNA Amplification." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/28118597521477734866.

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Анотація:

碩士
國立臺灣大學
電機工程學研究所
94
In this thesis, we develop a detecting method with high accuracy and high sensitivity for specific RNA sequences in isothermal environment. We construct a low-cost and real-time system for detection specific RNA sequences by using this method. Isothermal RNA amplification is a powerful tool to amplify specific sequences of RNA. It can successfully amplify as low as 1 ng of total RNA in 90 minutes. Therefore, we integrate isothermal RNA amplification and Fluorescence Resonance Energy Transfer (FRET) methods to real-time detect fluorescent signals for specific RNA sequences. For increase specificity, two hybridization probes which can bind to the same gene are used in our experiments to increase the accuracy. Base on this study, a novel method with higher accuracy and sensitivity for detecting specific RNA sequence in isothermal environment can be achieved. The results shown that the FRET signal of TRIM28 and UCHL1 genes have been detected in 40-60 min. Therefore, we have successfully setup a new low-cost, real-time detection system, based on combined Isothermal RNA Amplification and FRET.

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47

Goodrich, Terry T. "Ultrasensitive nucleic acid detection using surface plasmon resonance imaging measurements via a novel surface amplification process and microfluidic networks." 2004. http://www.library.wisc.edu/databases/connect/dissertations.html.

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48

Cox, Philipp [Verfasser]. "Etablierung und Evaluation der NASBA (Nucleic-acid-sequence-based-amplification)-Technologie zum Nachweis von Aspergillus-RNA / vorgelegt von Philipp Cox." 2006. http://d-nb.info/97814404X/34.

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49

Kosťun, Jan. "Využití molekulárně biologické metody One-Step Nucleic Acid Amplification (OSNA) při vyšetření sentinelových lymfatických uzlin u pacientek s karcinomem endometria." Doctoral thesis, 2018. http://www.nusl.cz/ntk/nusl-391393.

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Hypothesis The One-Step Nucleic Acid Amplification method could represent an effective intraoperative tool for detection of metastatic involvement of lymphatic nodes on the level of ultrastaging in endometrial cancer patients. Objective Utilization of the One-Step Nucleic Acid Amplification (OSNA) molecular biology method for the detection of the micrometastatic and macrometastatic involvement of sentinel lymph nodes in endometrial cancer patients. The objective is a comparison with the conclusion of the histopathological ultrastaging of sentinel lymph nodes and a description of the clinical consequences of this method. Methods Patients indicated for the surgical treatment of endometrial cancer underwent the detection of sentinel lymph nodes that was executed using the intracervical application of a tracer. Nodes larger than 5 mm were cut into sections 2 mm thick parallel to the short axis of the node. Odd sections were examined using the OSNA method, while even ones were examined by hematoxylin and eosin (H&E) and immunohistochemical examination to detect cytokeratin 19 antibody (IHC CK19) based on an ultrastaging-relevant protocol. Nodes of the size of 5 mm and smaller were divided into halves along the longitudinal axis with one half being examined using the OSNA method and the other half by...

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50

Omar, S. V. (Shaheed Vally). "Innovative approaches to tuberculosis diagnosis with emphasis on nucleic acid amplification tests in a resource constrained high burden tuberculosis setting." Thesis, 2015. http://hdl.handle.net/2263/50754.

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Анотація:

The global control of tuberculosis (TB) is currently hindered by the low sensitivity of microscopy and the prolonged time-to-result of culture. Recent technical progress has improved both diagnostic accuracy and turnaround, namely, nucleic acid amplification tests (NAAT). The World Health Organization (WHO) has recently endorsed two NAATs, which South Africa has been in the forefront of adopting. Based on WHO recommendations, the Xpert MTB/RIF assay (Xpert) has replaced microscopy as the firstline test in the National Algorithm. With current research and development primarily focused on rapid molecular tests, innovative methods of deployment are essential. In the work reported here, a contribution is offered towards fulfilling this need. This study aimed to show non-inferior diagnostic efficiency for the molecular detection of Mycobacterium tuberculosis from clinical sputum specimens in a novel specimen transport medium PrimeStore® - Molecular Transport Medium (PS-MTM). Technical evaluations of the parameters offered by the transport medium when applied to M. tuberculosis were performed; its ability to inactivate the organism, stabilize its deoxyribonucleic acid (DNA) in specimen over time and show compatibility with silica and magnetic bead-based DNA extraction systems for downstream molecular detection. Additionally, a novel and innovative sputum collection method, where a swab from sputum specimen placed into PS-MTM for the molecular detection of M. tuberculosis, is described. This collection system was evaluated in a routine clinical laboratory against mycobacterial culture, the reference standard. Collection method performance was further validated on sputum from suspected TB patients, at healthcare facilities in rural South Africa to a centralized laboratory for testing. Complete inactivation of M. tuberculosis occurred by 30 minutes after exposure, with a 1:3 sputum to PS-MTM ratio. The specimen remained stable with no significant change over time by real-time polymerase chain reaction (PCR) detection (<5% on a mean starting value) for PS-MTM samples over 28 days at ambient temperature. PS-MTM showed compatibility with all extraction systems; however, the automated bead-based extraction systems displayed better performance, with an estimated 170 CFU/ml lower limit of detection. Of 256 sputum specimens evaluated using the novel collection system, 10.2% were culture positive (routine specimen) and 11.0% positive by real-time PCR (PS-MTM swab from routine specimen). Against culture, detection of M. tuberculosis from swabbed sputum in PS-MTM had a sensitivity of 77% (CI 95%: 56-91%) and specificity of 96% (CI 95%: 93- 98%). Specimens obtained from 141 patients were included for the validation analysis, a subset of a larger cohort study. Concordance between the collection system under evaluation was 82% (McNemar, p=0.55) and 84% (McNemar, p=0.05) for culture and Xpert assay, respectively. Our findings suggest that PS-MTM is capable of improving safety and is an ideal solution for collecting, transporting and stabilizing sputum at ambient temperatures for centralized molecular TB testing. This system provides opportunities for resource-limited settings to introduce or further scale-up molecular diagnostics. PS-MTM samples are capable of bringing forward a significant number of positives, in addition to culture and Xpert testing, that could be regarded as real due to the system’s lower limits of detection and not just false-positives. Application of this system provides quality samples allowing for better discrimination, which in turn could provide adequate management of low bacillary load patients prior to transmission of infection.
Thesis (PhD)--University of Pretoria, 2015.
tm2015
Medical Microbiology
PhD
Unrestricted

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Дисертації з теми "Nucleic-acid Amplification and Quantification"

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