Department of Preservation and Food Safety Technologies, Institute of Agrochemistry and Food Technology, IATA-CSIC, Av. Agustín Escardino 7, Paterna, 46980, Valencia, Spain.
Department of Preservation and Food Safety Technologies, Institute of Agrochemistry and Food Technology, IATA-CSIC, Av. Agustín Escardino 7, Paterna, 46980, Valencia, Spain; Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, Burjassot, 46100, Valencia, Spain.
Sci Total Environ. 2021 Mar 1;758:143870. doi: 10.1016/j.scitotenv.2020.143870. Epub 2020 Dec 8.
Wastewater based epidemiology (WBE) has emerged as a reliable strategy to assess the coronavirus disease 2019 (COVID-19) pandemic. Recent publications suggest that SARS-CoV-2 detection in wastewater is technically feasible; however, many different protocols are available and most of the methods applied have not been properly validated. To this end, different procedures to concentrate and extract inactivated SARS-CoV-2 and surrogates were initially evaluated. Urban wastewater seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV) was used to test the concentration efficiency of an aluminum-based adsorption-precipitation method and a polyethylene glycol (PEG) precipitation protocol. Moreover, two different RNA extraction methods were compared in this study: a commercial manual spin column centrifugation kit and an automated protocol based on magnetic silica beads. Overall, the evaluated concentration methods did not impact the recovery of gamma-irradiated SARS-CoV-2 nor MgV, while extraction methods showed significant differences for PEDV. Mean recovery rates of 42.9 ± 9.5%, 27.5 ± 14.3% and 9.0 ± 2.2% were obtained for gamma-irradiated SARS-CoV-2, PEDV and MgV, respectively. Limits of detection (LoD) for five genomic SARS-CoV-2 targets (N1, N2, gene E, IP2 and IP4) ranged from 1.56 log genome equivalents (ge)/mL (N1) to 2.22 log ge/mL (IP4) when automated system was used; while values ranging between 2.08 (N1) and 2.34 (E) log ge/mL were observed when using column-based extraction method. Different targets were also evaluated in naturally contaminated wastewater samples with 91.2%, 85.3%, 70.6%, 79.4% and 73.5% positivity, for N1, N2, E, IP2 and IP4, respectively. Our benchmarked comparison study suggests that the aluminum precipitation method coupled with the automated nucleic extraction represents a method of acceptable sensitivity to provide readily results of interest for SARS-CoV-2 WBE surveillance.
基于污水的流行病学（WBE）已成为评估 2019 年冠状病毒病（COVID-19）大流行的可靠策略。最近的出版物表明，在污水中检测到 SARS-CoV-2 在技术上是可行的；然而，有许多不同的方案可用，而且大多数应用的方法都没有得到适当的验证。为此，最初评估了不同的程序来浓缩和提取失活的 SARS-CoV-2 和替代物。用伽马辐照的 SARS-CoV-2、猪流行性腹泻病毒（PEDV）和髓灰质炎病毒（MgV）接种的城市污水用于测试基于铝的吸附沉淀法和聚乙二醇（PEG）沉淀方案的浓缩效率。此外，本研究比较了两种不同的 RNA 提取方法：一种商用手动旋转柱离心试剂盒和一种基于磁性硅胶珠的自动化方法。总体而言，评估的浓缩方法对伽马辐照的 SARS-CoV-2 和 MgV 的回收没有影响，而提取方法对 PEDV 的回收有显著差异。获得的伽马辐照 SARS-CoV-2、PEDV 和 MgV 的平均回收率分别为 42.9±9.5%、27.5±14.3%和 9.0±2.2%。使用自动化系统时，针对 5 个 SARS-CoV-2 基因组靶点（N1、N2、基因 E、IP2 和 IP4）的检测限（LoD）范围为 1.56 log 基因组当量（ge）/mL（N1）至 2.22 log ge/mL（IP4）；而使用基于柱的提取方法时，观察到的范围在 2.08（N1）和 2.34（E）log ge/mL 之间。还评估了自然污染污水样本中的不同靶点，其阳性率分别为 N1、N2、E、IP2 和 IP4 的 91.2%、85.3%、70.6%、79.4%和 73.5%。我们的基准比较研究表明，铝沉淀法与自动化核酸提取相结合是一种具有可接受灵敏度的方法，可以为 SARS-CoV-2 WBE 监测提供有意义的快速结果。
