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

Abstract

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.

Real-Time On-Site Monitoring of Viruses in Wastewater Using Nanotrap® Particles and RICCA Technologies

“…Furthermore, the findings of the conventional quant istudio5 PRR machine demonstrated that the Nanotrap concentration method has a higher capability for collecting viruses in near-sensitivity proportion to the PEG technique. The findings of the investigation reveal that the Nanotrap microbiome particles effectively captured the virus to concentrate viral particles of SARS-CoV-2, present in wastewater, agreeing with previous studies [ 2 , 37 ]. Additionally, the use of a cellulose–acetate membrane-attached syringe filtering process, followed by a volume up from 10 to 30 mL, is also indicative of significant viral concentrations in wastewater samples via the use of a Nanotrap particle.…”

“…The concentration of SARS-CoV-2 was evaluated using two methods: (a) the Nanotrap magnetic virus particles (NMVP) method and (b) the PEG precipitation method [ 37 , 48 , 49 ]. To evaluate each concentration stage, a pre-treated aliquot of 50 mL was divided into two additional aliquots of 10 mL and 40 mL, each of which was then processed respectively.…”

“…The NMVP method used Nanotrap microbiome-A particles (SKU 44202-30, Ceres Nanosciences Inc., Manassas, VA, USA), which are magnetically functionalized affinity catch hydrogel nanoparticles that capture and concentrate biologically active microparticles [29,37]. In this procedure, 10 mL of the pre-treated aliquot in a sterile 15 mL conical falcon tube (Iwaki, Japan) was mixed with 100 µL of Nanotrap enhancement reagent-2 (ER2) (Ceres Nanoscience's SKU 10112-10) and gently shaken for 10 s. After that, 150 µL of Nanotrap microbiome particles (Ceres Nanoscience's SKU 44202-30) was added to the sample and mixed via gentle shaking.…”

“…Despite this, previous studies have highlighted various limitations, such as the viral recovery method, the sample source and volume, the absence of a nucleic acid extraction standard, and the limited primer and PCR testing kits [ 30 , 31 , 32 , 33 , 34 , 35 ]. Methods such as Polyethylene glycol (PEG) precipitation, skim milk flocculation, ultracentrifugation, and ultrafiltration are frequently utilized for concentrating SARS-CoV-2 in wastewater [ 36 , 37 , 38 , 39 ], and sophisticated laboratory conditions or equipment and specialized professionals are necessary for the quantitative monitoring of viruses in wastewater [ 40 , 41 , 42 , 43 ].…”

“…These particles capture and concentrate contaminants like viruses, bacteria, and live infectious pathogens from complex biological mixtures including blood, saliva, urine, and wastewater. The particles also help to separate contaminants from interfering components, capture numerous target molecules from complicated metrics of one sample, and preserve target molecules from degradation [ 37 , 44 ].…”