Antibiotic-resistant bacterial infections account for millions of human fatalities each year, with drug susceptibility testing (DST) affordability being limited by instrumentational constraints, monetary and/or time expenses. Molecular diagnostics performed at the point of care can provide a solution. Split probes coupled with label-free reporters like Fluorescent Light-up APtamers (FLAPs) are promising for point-of-care DST as they offer the needed selectivity towards point mutations inducive of drug resistance. This project aims at bridging the gap in FLAP applications for molecular diagnostics with a focus on multiplexing the analysis. Due to the limited number of DNA FLAPs available, we explored the ability of one of the most efficient DNA FLAPs - dapoxyl binding aptamer (DAP) - to bind fluorogens with different spectral properties. We performed the rational mutagenesis of the DAP dye-binding core to reveal any sequence-function correlations and to identify prospective orthogonal FLAP-dye pairs. Orthogonal FLAPs were used as scaffolds to design split dapoxyl aptameric (SDA) probes, which targeted a fragment of the katG gene from Mycobacterium tuberculosis complex associated with bacterial resistance to a first-line antituberculous drug isoniazid (INH). The probes were tested to differentiate the nucleic acid targets with single-nucleotide variations corresponding to isoniazid-susceptible (INHS) and isoniazid-resistant (INHR) bacterial phenotype in a multiplex fashion. With proper optimization of the probes, they can find an application in ratiometric analysis of heterogeneous bacterial populations composed of both drug-susceptible and drug-resistant strains and thus help in initial diagnosing of infectious diseases and in monitoring the therapy outcomes.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:honorstheses-2513 |
| Date | 01 January 2023 |
| Creators | Madalozzo, Pedro F |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Honors Undergraduate Theses |
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