Youssfi Wafa, Zhang Wen
Department of Civil Engineering, University of Arkansas, Fayetteville, Arkansas, USA.
Water Environ Res. 2025 Jul;97(7):e70146. doi: 10.1002/wer.70146.
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.
使用聚合酶链反应（PCR）对各种废水系统中的病毒载量进行量化的需求日益增加。本研究评估了两种常用工作流程：逆转录定量PCR（RT-qPCR）和逆转录液滴数字PCR（RT-ddPCR）在废水中的性能。我们通过测量添加到真实和合成废水样本中的模型病毒——铜绿假单胞菌噬菌体Φ6（Phi6）来比较这两种方法。真实污水取自阿肯色州费耶特维尔市西区污水处理设施的各个处理阶段。测试了包括从废水样本中提取RNA和不提取RNA的PCR在内的方案。据我们所知，这是第一项将模型病毒系统地添加到从多个处理阶段收集的废水中的研究，从而能够全面评估不同物理和化学特性基质中的病毒定量。研究结果表明，两种方法在检测高和中等病毒载量时表现出相似的性能。然而，RT-ddPCR对低病毒载量显示出显著更高的灵敏度，能够可靠地检测到RT-qPCR难以检测到的痕量病毒颗粒。对提取的废水样本进行RT-ddPCR测量也显示出对抑制剂的性能有所改善；然而，对于未提取RNA的样本，其检测受水质的影响更大。尽管RT-ddPCR成本更高、处理时间更长，但与RNA提取一起使用时，其卓越的灵敏度和对样本污染物的耐受性突出了其在废水应用中进行精确病毒监测的价值。
