Detection of SARS-CoV-2 and Other Viruses in Wastewater: Optimization and Automation of an Aluminum Hydroxide Adsorption–Precipitation Method for Virus Concentration

Abstract

This study aimed to provide a low-cost technique for virus detection in wastewater by improving an aluminum hydroxide adsorption–precipitation method. The releasing efficiency of viruses trapped by the aluminum hydroxide precipitates was improved by adding ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) to dissolve the precipitates at a Na 2 EDTA·2H 2 O:AlCl 3 molar ratio of 1.8–3.6. The recovery rates of the improved method for seven viruses, including SARS-CoV-2-abEN pseudovirus and six animal viruses, were 5.9–22.3% in tap water and 4.9–35.1% in wastewater. Rotavirus A (9.0–4.5 × 10 3 copies/mL), porcine circovirus type 2 (5.8–6.4 × 10 5 copies/mL), and porcine parvovirus (5.6–2.7 × 10 4 copies/mL) were detected in China’s pig farm wastewater, while rotavirus A (2.0 × 10 3 copies/mL) was detected in hospital wastewater. SARS-CoV-2 was detected in hospital wastewater (8.4 × 10 2 to 1.4 × 10 4 copies/mL), sewage (6.4 × 10 to 2.3 × 10 3 copies/mL), and river water (6.6 × 10 to 9.3 × 10 copies/mL) in Nepal. The method was automized, with a rate of recovery of 4.8 ± 1.4% at a virus concentration of 10 2 copies/mL. Thus, the established method could be used for wastewater-based epidemiology with sufficient sensitivity in coping with the COVID-19 epidemic and other virus epidemics.

Improving wastewater-based epidemiology performance through streamlined automation

“…Since each study calculates the LoD differently, the lowest detected viral load (RNA copies per mL of wastewater) is reported. Method Pre-Treatment Concentration Extraction Time* Recovery Efficiency** Lowest Detected Viral Load (copies/mL of wastewater) Adsorption –Precipitation [27] Manual Automated Manual 2 h 4.8±1.4% 64 Maxwell RSC RNA Extraction [28] Manual Manual Automated 3 h 30.2±17.7% 36 King Fisher Flex RNA Extraction [30] None Manual Automated 2.5 h 60.6±31.2% 80 King Fisher Flex RNA Extraction + Ceres Nanotrap ™ [29] Manual Automated Automated 3 h 27.0±8.0% ~100 Manual ESP [31] None Manual Manual 70 min 75.5±38.1% 206 Automated ESP None Automated Automated 40 min 84.5±25.4% 40 *The time it takes from raw wastewater to extracted RNA ready for PCR reaction. ** Average recovery efficiency over the reported range of viral concentrations.…”

“…Despite the inherent difficulty in automating key WBE processing steps, some promising progress has been made. For example, Yang et al developed a semi-automated aluminum hydroxide adsorption-precipitation method [27] , where solution is added to the sample and mixed mechanically and then filtered using the automated equipment. The precipitates are then collected using a manual scrapper and virus is eluted using EDTA-2Na dissolution.…”

Wastewater surveillance and an automated robot: effectively tracking SARS-CoV-2 transmission in the post-epidemic era

“…Briefly, 30 min centrifugation of 50 mL water sample was first applied to remove suspended solids (4°C, 2500 g) and then 0.5 mL of AlCl3 solution (0.3 mol/L) was added to the supernatant fluid with pH adjusted to 6.0 to allow aluminum hydroxide colloid emerging and adsorbing the virus. After that the pellet containing virus particles was separated by 5 min centrifugation (4°C, 1900 g) and 0.2 g EDTA-2Na was then mixed with the pellet to get higher releasing efficiency and to ensure the stability of the RNA [ 28 ].…”

Advances and implications of wastewater surveillance for SARS-CoV-2

“…沉淀法有絮凝沉降与聚乙二醇沉淀两种 机制. 前者主要通过使用絮凝剂(比如氯化铝)以中和病毒粒 子表面电荷形成聚集体, 然后再以乙二胺四乙酸二钠(EDTA-2Na)溶解胶体从而释放病毒颗粒, 具有成本低、适合自动 化、高通量等特点 [23] . 而聚乙二醇具有极强亲水性, 主要通 过结合溶液中水分子而使溶解的大分子物质析出沉淀 [24] .…”