Over the last 20 years, the incidence of Clostridium difficile infection (CDI) has increased dramatically, making it one of the most common healthcare-associated infections. This has been linked to the emergence of hypervirulent C. difficile strains. Currently, cell cytotoxicity assay (CTA) and toxigenic culture are the gold-standard methods for CDI diagnosis. However, they are time-consuming and labour-intensive. Other methods, like enzyme immunoassays (EIAs) and nucleic acid amplification-based tests (NAATs), allow for rapid testing but have poor sensitivity and/or specificity. Additionally, most of these methods target toxins or their associated genes and are unable to discriminate between epidemic and non-epidemic strains. The work described in this dissertation aims to develop easy-to-use and reliable biosensors for C. difficile, with a particular focus on epidemic strains of C. difficile. The development of the in vitro selection technique has allowed for the discovery of a big array of functional DNA, with excellent ability in both target recognition and enzymatic catalysis. My key interest is to employ functional DNA molecules as target recognition elements to develop colorimetric biosensors for C. difficile detection.
The first research project aimed to develop a colorimetric detection platform that can be coupled with functional DNA molecules to achieve hypersensitive detection of different targets. This test should be easy-to-use, have broad target applicability and not require expensive equipment. To do so, a colorimetric biosensing platform was created, which takes advantage of the signal amplification ability of rolling circle amplification (RCA) and the simplicity of the classic litmus test. In the presence of the target of interest, RCA will be triggered, and the biotinylated RCA products can hybridize a number of the urease-labelled single-stranded DNA and immobilize urease onto magnetic beads through streptavidin-biotin interactions. The urease can then be used to hydrolyze urea, resulting in significant pH elevation, that can be detected easily using a litmus test. To prove the concept, we have demonstrated that this platform can be employed to visually detect thrombin and platelet-derived growth factor (PDGF) with high sensitivity, by coupling it with an anti-thrombin aptamer and an anti-PDGF aptamer, respectively. We have also shown that the biosensing platform can be incorporated into simple paper-based devices.
The second project focuses on the development of a colorimetric DNA detection method for epidemic strains of C. difficile that utilizes both the polymerase chain reaction (PCR) and the litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in an increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of C. difficile, which are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients.
The goal of the third project was to isolate RNA-cleaving fluorogenic aptazymes (RFAs) targeting an epidemic strain of C. difficile. Four classes of RFA probes were derived using in vitro selection approach where a random-sequence DNA library was reacted with a crude extracellular mixture (CEM) derived from the epidemic C. difficile strain BI/027/NAP1, coupled with a subtractive selection strategy to eliminate cross-reactivities to unintended C. difficile strains and other bacterial species. The isolated RFDs can be used together to generate specific cleavage patterns for strain-specific identification of C. difficile.
Lastly, the final project was to characterize a novel RFA probe (RFA13-1) that was isolated unintentionally using in vitro selection. By using CEM prepared from C. difficile glycerol stock contaminated by Klebsiella aerogenes as the positive target for in vitro selection, we isolated a remarkably active RFA probe, RFA13-1, targeting K. aerogenes. Further studies demonstrated that RFA13-1 could be activated by CEM prepared from several bacteria from the Enterobacteriaceae family. Moreover, the molecular target of RFA13-1 has been identified, which is ribonuclease I. RFA13-1 showed high sensitivity and specificity towards RNase I and could be employed as a tool to study RNase I functions and to detect RNase I or RNase I-containing bacteria.
In summary, I have investigated novel strategies for building a biosensor that is capable of discriminately detecting epidemic strains of C. difficile. I hope that my work can take us one step closer towards the development of easy-to-use and reliable biosensors that can be used in the clinical diagnosis of CDI. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23769 |
| Date | January 2018 |
| Creators | Chang, Dingran |
| Contributors | Li, Yingfu, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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