Validating and optimizing the method for molecular detection and quantification of SARS-CoV-2 in wastewater

Area of Science:

• Environmental microbiology
• Molecular diagnostics
• Public health surveillance

Background:

• Wastewater surveillance is crucial for tracking SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) prevalence.
• Existing methodologies for SARS-CoV-2 detection in wastewater show significant variability.
• Standardized and validated methods are needed for reliable community infection estimation.

Purpose of the Study:

• To validate and optimize a molecular testing method for SARS-CoV-2 detection and quantification in wastewater.
• To improve the sensitivity and reliability of wastewater-based epidemiology for COVID-19.
• To establish a robust protocol for public health surveillance.

Main Methods:

• Concentration of viruses from wastewater using ultrafiltration.
• Optimization of sample storage (4°C), wastewater pH (9.6), and RNA extraction (MagMAX-96 viral RNA isolation kit).
• Detection using a one-step reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, with salmon DNA and pepper mild mottle virus (PMMoV) for inhibition monitoring and normalization.

Main Results:

• Storing wastewater samples at 4°C improved SARS-CoV-2 detection by 2-3 PCR-cycle threshold (Ct) values compared to -20°C.
• Adjusting wastewater pH to 9.6 enhanced virus recovery.
• The MagMAX-96 kit demonstrated the highest recovery rate for spiked human coronavirus strain 229E (hCoV-229E).
• One-step RT-qPCR showed improved sensitivity over two-step RT-qPCR for SARS-CoV-2 detection.

Conclusions:

• The validated ultrafiltration and one-step RT-qPCR method provides a sensitive and reliable tool for SARS-CoV-2 wastewater surveillance.
• Optimized sample handling and molecular assays enhance the accuracy of COVID-19 epidemiological data.
• This approach supports effective public health strategies through improved community infection monitoring.