Evaluation of Simple and Convenient Methods for SARS-CoV-2 Detection in Wastewater in high and Low Resource Settings

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA monitoring in wastewater has become an important tool for COVID-19 surveillance. Although many viral concentration methods such as membrane filtration and skim milk are reported, these methods generally require large volumes of wastewater, expensive lab equipment, and laborious processes. We utilized a Nanotrap(R) Microbiome A Particles (Nanotrap particle) method for virus concentration in wastewater. The method was evaluated across six parameters: pH, temperature, incubation time, wastewater volumes, RNA extraction methods, and two virus concentration approaches vs. a one-step method. The method was further evaluated with the addition of the Nanotrap Enhancement Reagent 1 (ER1) by comparing the automated vs. a manual Nanotrap particle method. RT-qPCR targeting the nucleocapsid protein was used for detection and quantification of SARS-CoV-2 RNA. Different pH, temperature, incubation time, wastewater volumes, and RNA extraction methods did not result in reduced SARS-CoV-2 detection in wastewater samples. The two-step concentration method showed significantly better results (P<0.01) than the one-step method. Adding ER1 to wastewater prior to viral concentration using the Nanotrap particles significantly improved PCR Ct results (P<0.0001) in 10 mL grab samples processed by automated Nanotrap particle method or 10 mL and 40 mL samples processed by manual Nanotrap particle method. SARS-CoV-2 detection in 10 mL grab samples with ER1 and the automated method showed significantly better (P=0.0008) results than 150 mL grab samples using the membrane filtration method. SARS-CoV-2 detection in 10 mL swab samples with ER1 via the automated method was also significantly better than without ER1 (P<0.0001) and the skim milk method in 250 mL Moore swab samples (P=0.012). These results suggest that Nanotrap methods could substitute the traditional membrane filtration and skim milk methods for viral concentration without compromising on the assay sensitivity. The manual method can be used in resource-limited areas, and the high-throughput platform is appropriate for large-scale COVID-19 wastewater-based surveillance.