Institute of Marine Sciences, University of North Carolina at Chapel Hill, Morehead City, North Carolina, United States.
Hampton Roads Sanitation District, Virginia Beach, Virginia, United States.
J Virol Methods. 2021 Nov;297:114230. doi: 10.1016/j.jviromet.2021.114230. Epub 2021 Jul 9.
Throughout the COVID-19 global pandemic there has been significant interest and investment in using Wastewater-Based Epidemiology (WBE) for surveillance of viral pathogen presence and infections at the community level. There has been a push for widescale implementation of standardized protocols to quantify viral loads in a range of wastewater systems. To address concerns regarding sensitivity, limits of quantification, and large-scale reproducibility, a comparison of two similar workflows using RT-qPCR and RT-ddPCR was conducted. Sixty raw wastewater influent samples were acquired from nine distinct wastewater treatment plants (WWTP's) served by the Hampton Roads Sanitation District (HRSD, Virginia Beach, Virginia) over a 6-month period beginning March 9th, 2020. Common reagents, controls, master mixes and nucleic acid extracts were shared between two individual processing groups based out of HRSD and the UNC Chapel Hill Institute of Marine Sciences (IMS, Morehead City, North Carolina). Samples were analyzed in parallel using One-Step RT-qPCR and One-Step RT-ddPCR with Nucleocapsid Protein 2 (N2) specific primers and probe. Influent SARS-CoV-2 N2 concentrations steadily increased over time spanning a range from non-detectable to 2.13E + 05 copies/L. Systematic dilution of the extracts indicated that inhibitory components in the wastewater matrices did not significantly impede the detection of a positive N2 signal for either workflow. The RT-ddPCR workflow had a greater analytical sensitivity with a lower Limit of Detection (LOD) at 0.066 copies/μl of template compared to RT-qPCR with a calculated LOD of 12.0 copies/μL of template. Interlaboratory comparisons using non-parametric correlation analysis demonstrated that there was a strong, significant, positive correlation between split extracts when employing RT-ddPCR for analysis with a ρ value of 0.86.
在整个 COVID-19 全球大流行期间，人们对使用基于废水的流行病学（WBE）来监测社区层面病毒病原体的存在和感染产生了浓厚的兴趣并进行了大量投资。已经有人推动广泛采用标准化协议来定量测量一系列废水系统中的病毒载量。为了解决关于灵敏度、定量下限和大规模重现性的问题，使用 RT-qPCR 和 RT-ddPCR 进行了两种类似工作流程的比较。在 2020 年 3 月 9 日开始的 6 个月期间，从由弗吉尼亚海滩汉普顿路污水处理区（HRSD，弗吉尼亚州）服务的 9 个不同污水处理厂（WWTP）采集了 60 个原始废水进水样本。HRSD 和北卡罗来纳州莫尔黑德州立大学海洋科学研究所（IMS）的两个独立处理组之间共享了常见的试剂、对照物、主混合物和核酸提取物。使用 One-Step RT-qPCR 和 One-Step RT-ddPCR 平行分析了使用核衣壳蛋白 2（N2）特异性引物和探针的样本。进水 SARS-CoV-2 N2 浓度随时间逐渐升高，范围从不可检测到 2.13E + 05 拷贝/L。系统稀释提取物表明，废水基质中的抑制性成分并没有显著阻碍两种工作流程中阳性 N2 信号的检测。RT-ddPCR 工作流程的分析灵敏度更高，检测限（LOD）为 0.066 拷贝/μl 模板，而 RT-qPCR 的计算 LOD 为 12.0 拷贝/μL 模板。使用非参数相关分析进行的实验室间比较表明，当使用 RT-ddPCR 进行分析时，分割提取物之间存在很强的、显著的正相关，ρ 值为 0.86。
