Comparison of Nanotrap(®) Microbiome A Particles, membrane filtration, and skim milk workflows for SARS-CoV-2 concentration in wastewater

INTRODUCTION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA monitoring in wastewater has become an important tool for Coronavirus Disease 2019 (COVID-19) surveillance. Grab (quantitative) and passive samples (qualitative) are two distinct wastewater sampling methods. Although many viral concentration methods such as the usage of membrane filtration and skim milk are reported, these methods generally require large volumes of wastewater, expensive lab equipment, and laborious processes. METHODS: The objectives of this study were to compare two workflows (Nanotrap(®) Microbiome A Particles coupled with MagMax kit and membrane filtration workflows coupled with RNeasy kit) for SARS-CoV-2 recovery in grab samples and two workflows (Nanotrap(®) Microbiome A Particles and skim milk workflows coupled with MagMax kit) for SARS-CoV-2 recovery in Moore swab samples. The Nanotrap particle workflow was initially evaluated with and without the addition of the enhancement reagent 1 (ER1) in 10 mL wastewater. RT-qPCR targeting the nucleocapsid protein was used for detecting SARS-CoV-2 RNA. RESULTS: Adding ER1 to wastewater prior to viral concentration significantly improved viral concentration results (P < 0.0001) in 10 mL grab and swab samples processed by automated or manual Nanotrap workflows. SARS-CoV-2 concentrations in 10 mL grab and Moore swab samples with ER1 processed by the automated workflow as a whole showed significantly higher (P < 0.001) results than 150 mL grab samples using the membrane filtration workflow and 250 mL swab samples using the skim milk workflow, respectively. Spiking known genome copies (GC) of inactivated SARS-CoV-2 into 10 mL wastewater indicated that the limit of detection of the automated Nanotrap workflow was ~11.5 GC/mL using the RT-qPCR and 115 GC/mL using the digital PCR methods. DISCUSSION: These results suggest that Nanotrap workflows could substitute the traditional membrane filtration and skim milk workflows for viral concentration without compromising the assay sensitivity. The manual workflow can be used in resource-limited areas, and the automated workflow is appropriate for large-scale COVID-19 wastewater-based surveillance.