Date of Graduation
5-2025
Document Type
Thesis
Degree Name
Master of Science in Civil Engineering (MSCE)
Degree Level
Graduate
Department
Civil Engineering
Advisor/Mentor
Zhang, Wen
Committee Member
Fairey, Julian L.
Second Committee Member
Guo, Lei
Keywords
Phi6; ribonucleic acid extraction.; RT-ddPCR; RT-qPCR; Wastewater
Abstract
There is an increasing demand for quantifying viral loads in diverse wastewater systems using polymerase chain reaction (PCR). This study evaluates the performance of two commonly used workflows: reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR) in wastewater. We compared the two methods by measuring the viral ribonucleic acid (RNA) of a model virus Phi6 in samples collected from various treatment stages at the Westside Wastewater Treatment Facility in Fayetteville, AR. RNA was extracted from real and synthetic wastewater samples and analyzed in parallel using both RT-qPCR and RT-ddPCR. Findings reveal that both methods demonstrated similar performance for detecting high and medium viral loads. However, RT-ddPCR showed significantly greater sensitivity for low viral loads, reliably detecting trace levels of viral particles where RT-qPCR struggled with detection. For direct viral detection without RNA extraction, RT-ddPCR's performance was more impacted by water quality, whereas measurement on extracted samples demonstrated improved performance against inhibitors. Although RT-ddPCR entails higher costs and longer processing times, its superior sensitivity and resilience to sample contaminants, when used with RNA extraction, underscore its value for precise viral monitoring in wastewater applications.
Citation
Youssfi, W. (2025). Comparison of RT-qPCR and RT-ddPCR on Assessing Model Virus in Wastewater. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/5659
