Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629, HZ, Delft, the Netherlands.
Technische Universität Darmstadt, Institute IWAR, 8 Franziska-Braun-Straße 7, 64287 Darmstadt, Germany.
Sci Total Environ. 2022 Jan 15;804:150244. doi: 10.1016/j.scitotenv.2021.150244. Epub 2021 Sep 10.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effects of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested: electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.
严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 大流行是人类最近面临的最困难挑战之一。基于污水的流行病学已成为监测和减轻潜在病毒爆发的一种工具，可以避免临床患者检测带来的偏差。由于情况紧急，从污水中分离病毒 RNA 所遵循的方案并未针对此类样本基质进行调整。在实施用于快速收集数据以维持监测和减轻决策的同时，分子方案需要协调一致，以提供准确、可重复和可比的分析结果。在这里，我们研究了与从污水中分离病毒 RNA 的方法相关的分析变异性。使用三种不同的进水污水体积来评估捕获病毒颗粒的过滤体积（50、100 或 500 mL）的影响。测试了三种不同的浓缩策略：负电膜、聚醚砜膜和阴离子交换二乙氨基乙基纤维素柱。为了比较病毒颗粒的数量，比较了不同的 RNA 分离方法（基于柱的方法与磁性珠方法）。还评估了额外的 RNA 纯化步骤和不同 RT-qPCR 策略（一步法与两步法）的影响。结果表明，当污水中存在基础病毒载量时，采用 500 mL 过滤体积通过负电膜和不进行多次 RNA 纯化步骤（使用基于柱的 RNA 纯化），并使用两步 RT-qPCR，避免了假阴性结果，并且在德尔夫特（荷兰海牙地区）进行的第二次浪潮监测期间产生了更一致的结果。通过为从污水中采样、浓缩和分子检测 SARS-CoV-2 病毒的方法标准化铺平道路，这些发现可以帮助水和卫生监测当局使用和信任来自基于污水的流行病学研究的结果，以便预测 SARS-CoV-2 爆发。
