Date of Graduation
5-2025
Document Type
Thesis
Degree Name
Bachelor of Science in Biomedical Engineering
Degree Level
Undergraduate
Department
Biomedical Engineering
Advisor/Mentor
Nelson, Christopher
Abstract
Pathogen contamination of irrigation systems and water sources threaten public health and agricultural infrastructure. Although PCR and other conventional nucleic acid detection methods are effective detection methods, their use of complex instrumentation and extensive sample processing limits field deployment in under-resourced and natural settings. This thesis evaluates Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), a CRISPR-Cas13a diagnostic tool, for ultrasensitive detection of viral genetic material in wastewater. SHERLOCK is able to maintain specificity via a custom CRISPR RNA (crRNA) and tolerance to common inhibitors. Plant and human viruses were simulated using a model system developed using a green fluorescent protein (GFP) gene target present in various forms. These targets were spiked into wastewater samples and then tested using SHERLOCK, which generates fluorescent signals by combining Cas13a collateral cleavage with isothermal pre-amplification. Viral nucleic acids were found even in complex wastewater matrices at low concentrations. Results showed clear fluorescent readout when comparing spiked lentiviral samples in wastewater to controls, consequently indicating SHERLOCK's potential use as a tool for wastewater pathogen detection. Next steps are modifying the system for bacterial and fungal pathogens and aggregating the assay into a field-deployable format.
Keywords
CRISPR-Cas13a; SHERLOCK; wastewater surveillance; viral detection; isothermal amplification; environmental diagnostics
Citation
Tolba, A. (2025). Detection of Viral Particles in Contaminated Wastewater Using CRISPR-Cas13a-SHERLOCK. Biomedical Engineering Undergraduate Honors Theses Retrieved from https://scholarworks.uark.edu/bmeguht/161
