Comparison of RT-qPCR and RT-ddPCR on Assessing Model Viruses in Wastewater

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 a model virus, Pseudomonas phage Φ6 (Phi6) spiked in both real and synthetic wastewater samples. Real sewage was collected from various treatment stages at the Westside Wastewater Treatment Facility in Fayetteville, AR. Protocols, including PCR with and without RNA extraction from wastewater samples, were tested. To the best of our knowledge, this is the first study to systematically spike a model virus into wastewater collected from multiple treatment stages, enabling a comprehensive assessment of viral quantification across matrices with distinct physical and chemical characteristics. 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. RT-ddPCR measurement on extracted wastewater samples also demonstrated improved performance against inhibitors; however, its detection was more impacted by water quality for samples without RNA extraction. Although RT-ddPCR entails higher costs and longer processing time, its superior sensitivity and resilience to sample contaminants when used with RNA extraction underscore its value for precise viral monitoring in wastewater applications.