Dimitrakopoulos Lampros, Kontou Aikaterini, Strati Areti, Galani Aikaterini, Kostakis Marios, Kapes Vasileios, Lianidou Evrikleia, Thomaidis Nikolaos, Markou Athina
Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, University Campus, Zografou, 15771, Athens, Greece.
Case Stud Chem Environ Eng. 2022 Dec;6:100224. doi: 10.1016/j.cscee.2022.100224. Epub 2022 Jun 23.
The ongoing pandemic caused by the emergence of SARS-CoV-2 has resulted in millions of deaths worldwide despite the various measures announced by the authorities. Wastewater-based epidemiology has the ability to provide a day-to-day estimation of the number of infected people in a fast and cost-effective manner. However, owing to the complex nature of wastewater, wastewater monitoring for viral genome copies is affected by the extensive viral fragmentation that takes place all the way to the sewage and the analytical lab. The aim of this study was to evaluate different methodologies for the concentration and extraction of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. We compare 5 different concentration methods and 4 commercially available kits for the RNA extraction. To evaluate the performance and the recovery of these, SARS-CoV-2 isolated from patients was used as a spike control. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and N3). Using spiked samples, recoveries were estimated 2.1-37.6% using different extraction kits and 0.1-2.1% using different concentration kits. It was found that a direct capture-based method, evaluated against a variety of concentration methods, is the best in terms of recovery, time and cost. Interestingly, we noticed a good agreement between the results provided by RT-qPCR and RT-ddPCR in terms of recovery. This evaluation can serve as a guide for laboratories establishing a protocol to perform wastewater monitoring of SARS-CoV-2. Overall, data presented here reinforces the validity of WBE for SARS-CoV-2 surveillance, uncovers potential caveats in the selection of concentration and extraction protocols and points towards optimal solutions to maximize its potential.
尽管当局宣布了各种措施，但由严重急性呼吸综合征冠状病毒2（SARS-CoV-2）出现引发的持续大流行已导致全球数百万人死亡。基于废水的流行病学能够以快速且经济高效的方式每日估算受感染人数。然而，由于废水的复杂性质，对病毒基因组拷贝的废水监测受到一直到污水和分析实验室发生的广泛病毒片段化的影响。本研究的目的是评估废水中病毒浓缩和提取的不同方法，并选择和改进一种能使SARS-CoV-2回收率最大化的方法。我们比较了5种不同的浓缩方法和4种市售的RNA提取试剂盒。为了评估这些方法的性能和回收率，将从患者分离出的SARS-CoV-2用作加标对照。此外，使用逆转录定量PCR（RT-qPCR）和逆转录液滴数字PCR（RT-ddPCR），针对三个基因标记（N1、N2和N3）测定所有废水样本中SARS-CoV-2的存在情况。使用加标样本，不同提取试剂盒的回收率估计为2.1%-37.6%，不同浓缩试剂盒的回收率估计为0.1%-2.1%。结果发现，与多种浓缩方法相比，基于直接捕获的方法在回收率、时间和成本方面是最佳的。有趣的是，我们注意到RT-qPCR和RT-ddPCR提供的回收率结果之间具有良好的一致性。该评估可为建立SARS-CoV-2废水监测方案的实验室提供指导。总体而言，此处呈现的数据强化了基于废水的流行病学用于SARS-CoV-2监测的有效性，揭示了浓缩和提取方案选择中的潜在问题，并指出了最大化其潜力的最佳解决方案。
