Quantifying Respiratory Viruses in WastewaterUsing GT Molecular’s Digital PCR Assays coupled with the CeresNanosciences Nanotrap® Workflow and QIAGEN AllPrep PowerViral™ Extraction Kit on the QIAcube™

KEY ADVANTAGES

• High-throughput multiplex detection coupling GT Molecular’s Digital PCR Assays with the Ceres Nanotrap Capture and Concentration Methods

• Ability to co-monitor and co-quantify multiple pathogens from a single sample

• Assays available for the QIAcuity® Platform

• Respiratory virus detection including: Influenza A, Influenza B and RSV in addition to SARS-CoV-2

INTRODUCTION

Monitoring the spread of SARS-CoV-2, the virus that causes COVID-19, as well as other pathogens such as RSV and Influenza continues to provide vital information for interrupting chains of transmission and preventing or dampening the spread of new cases. Wastewater testing continues to gain traction as a viable means to monitor pathogens and provide timely information about the changing prevalence within individual communities. Wastewater-based epidemiology (WBE) utility has already been successfully described to track infectious diseases like hepatitis, norovirus, and polio. Viral shedding detected in wastewater often precedes case upticks in healthcare settings, providing valuable and rapid information about infection within a community. Multiple countries have implemented wastewater-based monitoring of SARS-CoV-2, and the COVID-19 case rates highly correlate with SARS-CoV-2 RNA found in wastewater. However, these upper-respiratory viruses are often found at low concentrations within wastewater, thereby necessitating a concentration step. Ceres Nanosciences Nanotrap Particles, coupled with GT Molecular kits, provide end users high-throughput and sensitive means to monitor pathogens such as SARS-CoV-2, Influenza, and RSV.

METHODOLOGY

Automated Concentration and Extraction

Prior to sample processing, wastewater samples were first spiked with a known quantity of bovine coronavirus (BCoV) as an internal process control. Starting with 35-mL of wastewater from a de-identified location in Colorado, viral particles were first concentrated using the Ceres Nanotrap® Engancement Microbiome A particles and Enhancement Reagent 2. Automation for concentration was achieved using the KingFisher™ Apex instrument. Nucleic Acid was extracted using QIAGEN AllPrep PowerViral™ DNA/RNA Extraction kit on the QIAcube. Please note – an optimized heat step was incorporated during lysis. For step-by-step pro- tocol, please contact info@gtmolecular.com

Digital PCR

Concentrated wastewater extracts were subjected to digital PCR on the QIAGEN QIAcuity® platform us- ing GT Molecular dPCR assays. The N1 and N2 targets of SARS-CoV-2 as well as a bovine coronavirus (BCoV) internal control were quantified in a single reaction (GT Molecular catalog #100179). Influenza A, Influenza B, SARS-CoV-2 (SC2), and the same BCoV internal process control were quantified in a second reaction (GT Molecular catalog #100305).

Analysis

Data was analyzed using the QIAGEN QIAcuity® software. Control wells were first analyzed to ensure PCR ran efficiently and NTC wells were clean of contamination. Target quantification (copies/μL) was exported from the QIAcuity® software, and starting concentrations were back calculated based on the dilution factors achieved by the workflow. Quantification values were then imported into GraphPad Prism software and plotted.

RESULTS

Ceres Nanosciences and GT Molecular offer compatible workflows to monitor wastewater-borne SARS-CoV-2.

Human wastewater represents a complex, aggre- gate sample of an entire community. The detection of target pathogens from wastewater requires several steps prior to PCR. Specifically, pathogens must first be concentrated using methods that reduce the wastewater from milliliters to microliters. Digital PCR provides precise and reproducible quantification of genomic material in a sample. Kits include fecal indicator, pepper mild mosaic virus (PMMoV, data not shown). Concentrated RNA was subjected to dPCR with GT Molecular assays. As seen in Figure 1, SARS-CoV-2 was recovered and quantified. Importantly, viral recovery assessment was performed through the use of BCoV internal process control. This ensures final data accounts for viral loss and other wastewater variables including inhibitors.

Ceres Nanosciences and GT Molecular offer workflows to simultaneously monitor Influenza A, Influenza B, SARS-CoV-2, and RSV from a single wastewater sample.

Wastewater samples underwent the same proto- col described above, and the resulting wastewater extracts were subjected to dPCR to quantify SARS- CoV-2, Influenza A, and Influenza B. As seen in Figure 2, SARS-CoV-2 and Influenza A were recov- ered and quantified. Influenza B was not detected within the sample and mirrored local clinical epidemiology. Please note, GT Molecular offers an RSV add-on compatible with Catalog #100179 (Figure 1) or Catalog #100305 (Figure 2). This add-on allows users to achieve even further multiplexing and target monitoring within the same well as data collected above. All kits include fecal indicator pepper mild mosaic virus (PMMoV, data not shown).

DISCUSSION

Wastewater surveillance continues to gain traction as a non-invasive complement to traditional epi- demiology and offers an early-warning system for healthcare workers. Ceres Nanoscience technology provides a reliable and robust method to achieve ~350-fold increased concentration factor of a variety of microbes, proteins and nucleic acids. GT Molecular develops and validates digital PCR assays specifically for wastewater applications. The QIAGEN QIAcuity® dPCR platform affords simultaneous quantification of up to 5 targets within a single well. Collectively, the enclosed work outlines an automated, high-throughput, and end-to-end solution for users to quantify SARS-CoV-2, Influenza A, Influenza B, RSV, and required controls from a single wastewater sample.

CONCLUSION

This study shows that GT Molecular assays, when used with the AllPrep nucleic acid extraction protocol and Ceres Nanosciences Nanotrap technology can provide for surveillance of multiple pathogenic viruses from wastewater samples.

Email me the App Note!