Pathogenic bacteria pose serious threats to public health and safety. They can cause illness, death, and substantial economic losses. The most widely used bacterial detection methods include cell culturing, antibody-based assays, and nucleic acid amplification techniques, such as polymerase chain reaction (PCR). Unfortunately, these techniques are not well suited for point-of-care application, especially in the resource-limited regions of the world, as they require highly trained personnel to perform the test, they take a long time to complete (especially culturing), and they require sophisticated lab equipment. Thus, there is a great need for simpler, faster, and more accurate methods for bacterial detection. In this thesis, we present a simple, low-cost assay for detecting pathogenic bacteria that is based on the immobilization of a bacteria-specific RNA-cleaving DNAzyme (DNAzyme) onto a surface. If the target bacteria is present, a fluorescently labelled piece of DNA (FDNA) is released through the activity of the DNAzyme; if the target bacteria is not present, the FDNA remains attached to the surface as part of the DNAzyme construct. This method allows untrained users to determine whether a target bacteria is present by simply monitoring the fluorescence intensity in the liquid phase with a hand-held fluorimeter. The first step in this work was to experimentally evaluate different surfaces (including reduced graphene oxide and different beads) onto which the DNAzyme could be immobilized. These tests determined that agarose beads, covered with streptavidin, were ideally suited for DNAzyme immobilization. Next, we conducted a comparative evaluation of the kinetics/activity of the DNAzyme that had been immobilized onto the beads and the free DNAzyme in solution; the results of this evaluation revealed virtually identical reaction rates for the two cases, suggesting no loss of activity after immobilization. Finally, we explored how the DNAzyme sequence length influenced the assay. Specifically, we analyzed a full-length DNAzyme (Full DNAzyme) sequence and a truncated alternative (Short DNAzyme) and found that the full-length construct resulted in faster signal generation. Therefore, it was determined that the long version should be used in the assays.
When coupled with a filtration step, the immobilization of biotinylated DNAzymes onto the surface of streptavidin-coated agarose beads enabled the sensitive detection of E. coli in both water samples and complex matrices, such as milk and apple juice. The bead-based assay was able to produce a strong fluorescence signal readout in as little as 2.5 min following contact with E. coli, and it was capable of achieving a detection limit of 1,000 colony-forming units (CFUs) without sample enrichment. As DNAzyme probes can be generated through in vitro selection to react to different bacteria, the RNA-cleavage based detection mechanism described in this work can be adapted for the detection of a wide range of bacterial targets. Overall, this research has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that is highly attractive for field applications, especially in resource-limited regions. / Thesis / Master of Applied Science (MASc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23994 |
| Date | January 2019 |
| Creators | Esmaeili Samani, Sahar |
| Contributors | Filipe, Carlos, Li, Yingfu, Chemical Engineering |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0018 seconds