The SARS-CoV-2 pandemic has accelerated the development of virus concentration and molecular-based virus detection methods, monitoring systems and overall approach to epidemiology. Early into the pandemic, wastewater-based epidemiology started to be employed as a tool for tracking the virus transmission dynamics in a given area. The complexity of wastewater coupled with a lack of standardized methods led us to evaluate each step of the analysis individually and see which approach gave the most robust results for SARS-CoV-2 monitoring in wastewater. In this article, we present a step-by-step, retrospective view on the method development and implementation for the case of a pilot monitoring performed in Slovenia. We specifically address points regarding the thermal stability of the samples during storage, screening for the appropriate sample concentration and RNA extraction procedures and real-time PCR assay selection. Here, we show that the temperature and duration of the storage of the wastewater sample can have a varying impact on the detection depending on the structural form in which the SARS-CoV-2 target is present. We found that concentration and RNA extraction using Centricon filtration units coupled with Qiagen RNA extraction kit or direct RNA capture and extraction using semi-automated kit from Promega give the most optimal results out of the seven methods tested. Lastly, we confirm the use of N1 and N2 assays developed by the CDC (USA) as the best performing assays among four tested in combination with Fast Virus 1-mastermix. Data show a realistic overall process for method implementation as well as provide valuable information in regards to how different approaches in the analysis compare to one another under the specific conditions present in Slovenia during a pilot monitoring running from the beginning of the pandemic.

SARS-CoV-2 大流行加速了病毒浓度和基于分子的病毒检测方法、监测系统和流行病学整体方法的发展。在大流行初期，基于废水的流行病学开始被用作跟踪特定区域病毒传播动态的工具。废水的复杂性加上标准化方法的缺乏使我们单独评估分析的每个步骤，看看哪种方法为废水中的 SARS-CoV-2 监测提供了最可靠的结果。在本文中，我们针对在斯洛文尼亚进行的试点监测案例，展示了方法开发和实施的循序渐进的回顾性观点。我们专门解决了样品在储存过程中的热稳定性问题，筛选合适的样品浓度和 RNA 提取程序以及实时 PCR 检测选择。在这里，我们表明，根据 SARS-CoV-2 靶标存在的结构形式，废水样本的温度和储存时间会对检测产生不同的影响。我们发现，使用 Centricon 过滤装置和 Qiagen RNA 提取试剂盒进行浓缩和 RNA 提取，或使用 Promega 的半自动试剂盒直接捕获和提取 RNA，在七种测试方法中给出了最佳结果。最后，我们确认使用由 CDC（美国）开发的 N1 和 N2 检测作为与 Fast Virus 1-mastermix 组合测试的四种检测中性能最好的检测。