Globally, tuberculosis, a disease caused by the species of Mycobacterium tuberculosis (Mtb) complex, stands as a leading cause of death from a single infectious agent. Even though antituberculous drugs are available, treatment is challenging due to antibiotic resistance associated with point mutations in the bacterial genome. Resistance to the first-line antibiotics – rifampin and isoniazid – results in multidrug-resistant tuberculosis (MDR) requiring a more complicated treatment regimen. Timely and accurate identification of drug-resistant TB cases can help prescribe the most effective treatment and prevent the spread of infection. This research aims to develop an assay to discern multi-drug resistant forms of tuberculosis using a molecular assay based on split deoxyribozyme hybridization probes. For the probe design, a catalytic core of an RNA-cleaving deoxyribozyme is split into two parts, with each part elongated with a target-recognizing fragment ("arm"). In the presence of a fully complementary nucleic acid target, but not the one containing point mutations, the catalytic core of the deoxyribozyme can be re-formed due to the assembling of the target-probe complex, which recognizes and allows cleavage of a fluorophore- and quencher-labeled signal reporter, thereby ensuring increase in fluorescence in a target-dependent manner. The target-binding arms of the probes were optimized in terms of the signal-to-background ratio and selectivity of target recognition using synthetic targets corresponding to the fragments of the katG and rpoB genes with point-mutation sites implicated in the resistance to isoniazid and rifampin, respectively. The optimized probe sequences were used to interrogate the targets obtained by amplifying the correspondent fragments of the Mtb genes using Linear- After-The-Exponential (LATE) PCR, which allows efficient synthesis of a single-stranded amplicon. The signal triggered by cognate targets can be read using a portable fluorometer, which eliminates the need to use a sophisticated real-time PCR instrument for the assay. The success of the split deoxyribozyme assay can establish an affordable and user-friendly molecular diagnostic assay where a sample can be amplified and analyzed in a single tube.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd2020-2558 |
| Date | 01 January 2023 |
| Creators | Fergus, Abryana |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations, 2020- |
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