Methods Evaluation for Rapid Concentration and Quantification of SARS-CoV-2 in Raw Wastewater Using Droplet Digital and Quantitative RT-PCR.
Methods Evaluation for Rapid Concentration and Quantification of SARS-CoV-2 in Raw Wastewater Using Droplet Digital and Quantitative RT-PCR.
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DOI：
10.1007/s12560-021-09488-8
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发表时间：
2021-09
影响因子：
3.4
通讯作者：
Aw TG
中科院分区：
文献类型：
--
作者：
Flood MT;D'Souza N;Rose JB;Aw TG
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的废水监测是一种新兴的公共卫生工具，用于了解2019冠状病毒病（COVID-19）在社区中的传播。需要评估不同病毒浓缩方法和PCR方法的性能，以确定其是否适用于检测废水中的SARS-CoV-2。我们评估了超滤和聚乙二醇（PEG）沉淀方法，以浓缩污水处理厂和污水网络上游（例如，人孔、升降机站）。使用Phi6噬菌体作为包膜病毒的替代物，通过不同浓度方法测定病毒的回收率。此外，使用逆转录定量PCR（RT-qPCR）和逆转录液滴数字PCR（RT-ddPCR）确定所有废水样品中SARS-CoV-2的存在，靶向三个遗传标记（N1，N2和E）。使用加标样品，使用基于超滤的方法估计Phi6回收率为2.6 - 11.6%，使用PEG沉淀法估计为22.2 - 51.5%。在PEG程序中，有和没有16小时过夜孵育之间的回收效率没有显著差异（p <0.05），证明了获得当天结果的可行性。RT-ddPCR检测SARS-CoV-2遗传标记的灵敏度和精密度均高于RT-qPCR。虽然所有三个SARS-CoV-2的遗传标记，使用RT-ddPCR检测，E基因的水平几乎低于检测限使用RT-qPCR。总的来说，我们的研究表明PEG沉淀是一种有效的低成本方法，可以在SARS-CoV-2的常规废水监测中同时处理大量样品。RT-ddPCR可用于不同废水基质中SARS-CoV-2遗传标记的绝对定量。在线版本包含补充材料，可通过10.1007/s12560 - 021 - 09488 - 8获取。
Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging public health tool to understand the spread of Coronavirus Disease 2019 (COVID-19) in communities. The performance of different virus concentration methods and PCR methods needs to be evaluated to ascertain their suitability for use in the detection of SARS-CoV-2 in wastewater. We evaluated ultrafiltration and polyethylene glycol (PEG) precipitation methods to concentrate SARS-CoV-2 from sewage in wastewater treatment plants and upstream in the wastewater network (e.g., manholes, lift stations). Recovery of viruses by different concentration methods was determined using Phi6 bacteriophage as a surrogate for enveloped viruses. 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 E). Using spiked samples, the Phi6 recoveries were estimated at 2.6–11.6% using ultrafiltration-based methods and 22.2–51.5% using PEG precipitation. There was no significant difference in recovery efficiencies (p < 0.05) between the PEG procedure with and without a 16 h overnight incubation, demonstrating the feasibility of obtaining same day results. The SARS-CoV-2 genetic markers were more often detected by RT-ddPCR than RT-qPCR with higher sensitivity and precision. While all three SARS-CoV-2 genetic markers were detected using RT-ddPCR, the levels of E gene were almost below the limit of detection using RT-qPCR. Collectively, our study suggested PEG precipitation is an effective low-cost procedure which allows a large number of samples to be processed simultaneously in a routine wastewater monitoring for SARS-CoV-2. RT-ddPCR can be implemented for the absolute quantification of SARS-CoV-2 genetic markers in different wastewater matrices. The online version contains supplementary material available at 10.1007/s12560-021-09488-8.
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影响因子：
4
作者：
Aw, T. G.;Gin, K. Y-H.
通讯作者：
Gin, K. Y-H.
DOI：
10.1016/j.scitotenv.2020.143101
发表时间：
2021-03-25
期刊：
The Science of the total environment
影响因子：
--
作者：
Cuevas-Ferrando E;Pérez-Cataluña A;Allende A;Guix S;Randazzo W;Sánchez G
通讯作者：
Sánchez G
影响因子：
9.8
作者：
Ahmed, Warish;Bertsch, Paul M.;Kitajima, Masaaki
通讯作者：
Kitajima, Masaaki
影响因子：
3.4
作者：
Blanco, Albert;Abid, Islem;Bosch, Albert
通讯作者：
Bosch, Albert
影响因子：
5.2
作者：
Gendron, Louis;Verreault, Daniel;Duchaine, Caroline
通讯作者：
Duchaine, Caroline