Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.705555
Title: Developing new synthetic tools for nucleic acid based diagnostics
Author: Xu, Gaolian
ISNI:       0000 0004 6060 5239
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2016
Availability of Full Text:
Access through EThOS:
Full text unavailable from EThOS. Thesis embargoed until 01 Oct 2018
Access through Institution:
Abstract:
With increasing globalization, new infectious diseases are being discovered and spread quickly around the world. The traditional ways for the detection and identification of infectious pathogens are time-consuming and usually require specific facilities. This may delay effective treatment and lead to the spread of infectious disease, especially in the early stages of an epidemic. Therefore, accurate and efficient methods for identification of causative pathogens are very important. Here, we take advantages of new synthetic tools and nucleic acid technologies to develop novel infectious disease diagnostic methods that are user-friendly and have high sensitivity and specificity. These include: 1). The development of a rapid ultrasonic DNA isothermal amplification method with multiplexed melting analysis; 2). The development of an origami device based nucleic acid multiplexed detection method; 3). The development of a novel branched Hybridization Chain Reaction (HCR) assay. 1. Rapid ultrasonic isothermal amplification of DNA with multiplexed melting analysis-applications in the clinical diagnosis of sexually transmitted diseases (1) In this project, surface acoustic wave (SAW) signals are generated by interdigitated transducer (IDT) on LiNbO3 and propagated into disposable silicon superstrates on which a droplet of Loop-mediated isothermal amplification (LAMP) reaction mixture has been placed. As SAW interacts with the LAMP mixture, its energy is transferred into the LAMP mixture and causes the temperature of the mixture to increase. By controlling the SAW generation voltage, the temperature of the LAMP mixture could be maintained within a certain range as necessary for the LAMP reaction. During the process of LAMP amplification, a lot of double-strand DNA (dsDNA) is produced; this can incorporate specific fluorescent dyes and result in an exponential increase in fluorescence signal intensity. Also, by gradually increasing the SAW generation voltage, a SAW actuation-based DNA melting method could be used for multiplex detection. Ten-fold serially diluted targets from 105 copies/reaction to 10 copies/reaction were used to quantify the analytical sensitivity of the SAW-LAMP system and measurable signals were found down to 10 copies/reaction. Compared to a Peltier-LAMP system, SAW actuation enables the amplification to be performed more rapidly, about 18.23 % +/- 2.5 faster. Six clinical samples were used to demonstrate the clinical validation of SAW-LAMP by comparing with results from qPCR. The use of a SAW actuation-based DNA melting method distinguished the difference between melting temperatures of C. trachomatis amplicon (79.65 +/- 0.14 °C), and N. gonorrhoea (82.55 +/- 0.53 °C). 2. Development of paper origami device based nucleic acid multiplex detection for infectious diseases diagnostics In this project, a paper-folding origami device to manipulate malaria-infected blood samples was described. Through the simple process of paper folding, the nucleic acid of parasites in the blood sample could be extracted, purified and eluted. The extracted nucleic acid was then amplified with a multiplexed colorimetric LAMP assay in a plastic plate. Finally, the amplification products of multiplexed colorimetric LAMP assay were detected within an array with a low cost hand-held torch by naked eye. The multiplexed colorimetric LAMP assays for Plasmodium pan, Plasmodium falciparum, Plasmodium vivax with an internal control (IC) were investigated. The analytical sensitivity of colorimetric LAMP assays was tested by WHO International Standard DNA, with the limit of detection down to 105 IU/ml. Serially diluted quantified hCMV genomic DNA was used to demonstrate the DNA recovery of our origami device, which was between 60-70%. Serial dilution of a known infected blood samples (from 100 parasites/μl to 1 parasites/μl) were used to study the analytical sensitivity and obtain an LOD of the origami device to 5 parasites/μl. 80 fully characterised fresh malaria infected blood samples were used to assess the clinical validation effect of our origami device through a double blind, randomized controlled, clinical trial. All samples were also tested with commercially available LAMP kit and benchmark real-time PCR assay. The coincidence of our method and benchmark PCR were 88.75% (71/80) for Plasmodium pan, 90% (72/80) for P. falciparum and 93.75% (75/80) for P. vivax. Similarly, the coincidence between our method and the LAMP kit for Plasmodium pan and P. falciparum were 90% (72/80) and 92.50% (74/80) respectively. Using benchmark PCR as a gold standard for the detection of Plasmodium pan, P. ovale, P. falciparum and P. vivax, the sensitivity for our tests was of 85.5%, 92.9%, 61.1% and 81.0%, respectively. While the specificity are 100%, 94.2%, 98.5% and 98.30% respectively. We also established our origami device can diagnose species type from stored samples (either frozen, fixed, or dried). 3. Development of a novel branched Hybridization chain reaction (HCR) By increasing the dimensionality of an HCR system, a novel branched HCR product with complex branched structures instead of linear constructs has been developed. To validate the principle of a transition from a 1D chain to a higher dimension, we adapted a 3-arm branching construct to enable it to form a chain reaction by incorporating hybridization tails onto its sequences. The novel branched HCR reaction can form three-arm junction units with the introduction of a specific initiator. The three-armed units formed not only freed initiators to start of another cycle of HCR, but also bonded to each other to form complex and branched products. We also show that the highly branched polymers produced allow label-free acoustic mass sensing. The product of branched HCR was detected by Love Wave (LW) biosensor with the limit of detection at 2 nM, meaning 0.1% - 0.2% working frequency shift. Based on the branched HCR, we also design a new multiplexed HCR mechanism, where a single reaction is able to detect the presence of different initiators. It is based on designing primers that carry additional hairpin structures, which cross-react specifically upon initiation, yielding branching, thus opening up new applications for this enzyme-free, label-free DNA detection system.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.705555  DOI: Not available
Keywords: RA0421 Public health. Hygiene. Preventive Medicine ; RZ Other systems of medicine
Share: