Characterization and Optimization of Nanotrap® Microbiome Particlemethods for Concentrating SARS-CoV-2 from Small Volume Wastewater Samples

SARS-CoV-2 RNA monitoring in wastewater has become an important tool for COVID-19 surveillance. Although many viral concentration methods are reported, these methods generally require large volumes of wastewater, expensive lab equipment, and laborious processes. We utilized a Nanotrap® Microbiome A Particles (briefly 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). Using Nanotrap particles and ER1 on the automated system or using the manual Nanotrap particles method and ER1 enable rapid, sensitive detection of SARS-CoV-2 in 10 mL wastewater samples. 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.