Akter Jesmin, Smith Wendy J M, Liu Yawen, Kim Ilho, Simpson Stuart, Thai Phong, Korajkic Asja, Ahmed Warish
Civil and Environmental Engineering, University of Science and Technology (UST), Daejeon 34113, Republic of Korea; Department of Environmental Research, Korea Institute of Civil Engineering and Building Technology (KICT), Gyeonggi-do 10223, Republic of Korea; CSIRO Environment, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia.
CSIRO Environment, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia.
Sci Total Environ. 2023 Oct 27:167966. doi: 10.1016/j.scitotenv.2023.167966.
The lack of standardized methods and large differences in virus concentration and extraction workflows have hampered severe acute respiratory syndrome (SARS-CoV-2) wastewater surveillance and data reporting practices. Numerous studies have shown that adsorption-extraction (AE) method holds promise, yet several uncertainties remain regarding the optimal AE workflow. Several procedural components that may influence the recovered concentrations of target DNA/RNA, including membrane types, homogenization instruments, speed and duration, and lysis buffer. In this study, 42 different AE workflows that varied these components were compared to determine the optimal method by quantifying endogenous SARS-CoV-2, human adenovirus (HAdV 40/41) and a bacterial marker gene of fecal pollution (Bacteroides HF183). Our findings suggest that the certain selected workflow had a significant impact on SARS-CoV-2 concentrations, whereas it had minimal impact on HF183 and no effect on HAdV 40/41 concentrations. When comparing individual components in a workflow, such as membrane type (MF-Millipore™ 0.45 μm MCE vs. Isopore™ 0.40 μm) and homogenization instruments (Precellys 24 homogenizer vs. Vortex-Genie®-2), we found that they had no impact on SARS-CoV-2, HAdV 40/41, and HF183 concentrations. This suggests that at least some consumables and equipment are interchangeable. Buffer PM1 + TRIzol based workflows yielded higher concentrations of SARS-CoV-2 than other workflows. HF183 concentrations were higher in workflows without chloroform. Similarly, higher homogenization speeds (5000-10,000 rpm) led to increased concentrations of SARS-CoV-2 and HF183 but had no effect on HAdV 40/41. Our findings indicate that minor enhancements to the AE workflow can improve the recovery of viruses and bacteria from the wastewater, leading to different outcomes from wastewater surveillance efforts.
缺乏标准化方法以及病毒浓度和提取流程存在巨大差异，阻碍了严重急性呼吸综合征冠状病毒2（SARS-CoV-2）废水监测及数据报告工作。众多研究表明，吸附提取（AE）方法具有潜力，但关于最佳AE流程仍存在一些不确定性。几个可能影响目标DNA/RNA回收浓度的程序组件，包括膜类型、匀浆仪器、速度和持续时间以及裂解缓冲液。在本研究中，比较了42种改变这些组件的不同AE流程，通过定量内源性SARS-CoV-2、人腺病毒（HAdV 40/41）和粪便污染的细菌标记基因（拟杆菌HF183）来确定最佳方法。我们的研究结果表明，某些选定的流程对SARS-CoV-2浓度有显著影响，而对HF183影响最小，对HAdV 40/41浓度无影响。在比较流程中的各个组件时，如膜类型（密理博™ 0.45μm MCE膜与等孔™ 0.40μm膜）和匀浆仪器（Precellys 24匀浆器与Vortex-Genie®-2涡旋振荡器），我们发现它们对SARS-CoV-2、HAdV 40/41和HF183浓度均无影响。这表明至少一些耗材和设备是可互换的。基于缓冲液PM1 + 氯仿异硫氰酸胍的流程比其他流程产生更高浓度的SARS-CoV-2。在没有氯仿的流程中HF183浓度更高。同样，更高的匀浆速度（5000 - 10000转/分钟）导致SARS-CoV-2和HF183浓度增加，但对HAdV 40/41无影响。我们的研究结果表明，对AE流程进行微小改进可以提高废水中病毒和细菌的回收率，从而使废水监测工作产生不同的结果。
