Environmental Microbiome Engineering and Biotechnology Laboratory, Center for Environmental Engineering Research, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam Road, Hong Kong, China; HKU-Pasteur Research Pole, Pokfulam Road, Hong Kong, China.
Sci Total Environ. 2022 Jun 10;824:153687. doi: 10.1016/j.scitotenv.2022.153687. Epub 2022 Feb 5.
Wastewater surveillance is a promising tool for population-level monitoring of the spread of infectious diseases, such as the coronavirus disease 2019 (COVID-19). Different from clinical specimens, viruses in community-scale wastewater samples need to be concentrated before detection because viral RNA is highly diluted. The present study evaluated eleven different virus concentration methods for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater. First, eight concentration methods of different principles were compared using spiked wastewater at a starting volume of 30 mL. Ultracentrifugation was the most effective method with a viral recovery efficiency of 25 ± 6%. The second-best option, AlCl precipitation method, yielded a lower recovery efficiency, only approximately half that of the ultracentrifugation method. Second, the potential of increasing method sensitivity was explored using three concentration methods starting with a larger volume of 1000 mL. Although ultracentrifugation using a large volume outperformed the other two large-volume methods, it only yielded a comparable method sensitivity as the ultracentrifugation using a small volume (30 mL). Thus, ultracentrifugation using less volume of wastewater is more preferable considering the sample processing throughput. Third, a comparison of two viral RNA extraction methods showed that the lysis-buffer-based extraction method resulted in higher viral recovery efficiencies, with cycle threshold (Ct) values 0.9-4.2 lower than those obtained for the acid-guanidinium-phenol-based method using spiked samples. These results were further confirmed by using positive wastewater samples concentrated by ultracentrifugation and extracted separately by the two viral RNA extraction methods. In summary, concentration using ultracentrifugation followed by the lysis buffer-based extraction method enables sensitive and robust detection of SARS-CoV-2 for wastewater surveillance.
污水监测是一种用于监测传染病（如 2019 年冠状病毒病（COVID-19））在人群中传播的有前途的工具。与临床标本不同，社区规模污水样本中的病毒在检测之前需要浓缩，因为病毒 RNA 高度稀释。本研究评估了 11 种不同的病毒浓缩方法，用于检测污水中的严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）。首先，使用起始体积为 30 mL 的加标污水比较了 8 种不同原理的浓缩方法。超速离心是最有效的方法，病毒回收率为 25±6%。效果次之的是 AlCl 沉淀法，回收率仅约为超速离心法的一半。其次，使用三种起始体积为 1000 mL 的浓缩方法探索了提高方法灵敏度的潜力。尽管使用大体积进行超速离心的效果优于其他两种大体积方法，但与使用小体积（30 mL）进行超速离心的方法灵敏度相当。因此，考虑到样品处理通量，使用较少体积的污水进行超速离心更为可取。第三，两种病毒 RNA 提取方法的比较表明，基于裂解缓冲液的提取方法可获得更高的病毒回收率，与基于酸胍-苯酚的方法相比，加标样品的 Ct 值低 0.9-4.2。使用超速离心浓缩的阳性污水样本，并分别使用两种病毒 RNA 提取方法进行提取，进一步证实了这一结果。总之，使用超速离心浓缩，然后使用基于裂解缓冲液的提取方法，可实现污水监测中 SARS-CoV-2 的敏感和稳健检测。
