Green Ashley, Pappu Aiswarya Rani, Oakes Melanie, Sandmeyer Suzanne, Hileman Matthew, Jiang Sunny
Department of Civil and Environmental Engineering, University of California Irvine, Irvine, CA 92697, USA.
Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA.
Microorganisms. 2025 Aug 9;13(8):1862. doi: 10.3390/microorganisms13081862.
Wastewater can serve as both a source of pathogens that pose risks to human health and a valuable resource for tracking and predicting disease prevalence through wastewater-based surveillance (WBS). In WBS for SARS-CoV-2, both nucleocapsid-specific (N1 and N2) and the envelope (E) genes are common targets for primer design, but ambiguity remains regarding differences in results depending on the gene target chosen. This study investigated how and why two SARS-CoV-2 gene targets (N2 and E) varied when analyzed in a multiplex RT-ddPCR assay for a COVID-19 wastewater monitoring study. From December 2021 to June 2022, over 700 raw wastewater samples were collected from thirteen manholes in the University of California, Irvine sewer system. Murine hepatitis virus (MHV) was used as a matrix recovery and process control in the triplex RT-ddPCR assay. Water quality tests (TSS, COD, pH, turbidity and NH-N) were performed on all samples. Analyses showed that in over 10% of samples, the E gene concentration exceeded N2 by more than one order of magnitude. To evaluate matrix effects on amplification efficiency for N2 and E genes, multiple regression analysis was performed to explore whether water quality variables and MHV recovery efficiency could predict variance in gene concentrations, but no clear relationship was identified. However, viral recovery, as indicated by MHV recovery efficiency, was negatively impacted in samples with higher TSS and COD, suggesting PCR inhibition. These findings contribute to methodological standardization efforts in WBS and emphasize the importance of primer selection for large-scale monitoring.
废水既可能是对人类健康构成风险的病原体来源，也可作为通过基于废水的监测（WBS）来追踪和预测疾病流行情况的宝贵资源。在针对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的WBS中，核衣壳特异性（N1和N2）基因以及包膜（E）基因都是引物设计的常见靶点，但对于根据所选基因靶点得出的结果差异，仍存在不确定性。本研究调查了在一项用于2019冠状病毒病废水监测研究的多重逆转录数字滴液聚合酶链反应（RT-ddPCR）检测中，两种SARS-CoV-2基因靶点（N2和E）在分析时如何以及为何会有所不同。在2021年12月至2022年6月期间，从加利福尼亚大学欧文分校下水道系统的13个检修孔采集了700多个未经处理的废水样本。在三重RT-ddPCR检测中，鼠肝炎病毒（MHV）用作基质回收率和过程控制。对所有样本进行了水质检测（总悬浮固体、化学需氧量、pH值、浊度和氨氮）。分析表明，在超过10%的样本中，E基因浓度超过N2基因浓度一个以上数量级。为了评估基质对N2和E基因扩增效率的影响，进行了多元回归分析，以探讨水质变量和MHV回收率是否能够预测基因浓度的差异，但未发现明确的关系。然而，如MHV回收率所示，病毒回收率在总悬浮固体和化学需氧量较高的样本中受到负面影响，表明存在聚合酶链反应抑制现象。这些发现有助于WBS的方法标准化工作，并强调了引物选择对大规模监测的重要性。
