Evaluation of multiple analytical methods for SARS-CoV-2 surveillance in wastewater samples

Area of Science:

• Environmental microbiology
• Virology
• Public health surveillance

Background:

• Wastewater surveillance is crucial for monitoring infectious diseases like SARS-CoV-2.
• Optimizing virus concentration and RNA extraction is essential for accurate SARS-CoV-2 quantification in wastewater.

Purpose of the Study:

• To evaluate and compare 50 different protocols for SARS-CoV-2 concentration and RNA extraction from wastewater.
• To identify the most efficient method for SARS-CoV-2 detection and quantification in wastewater samples.
• To assess the impact of prolonged storage on enveloped virus RNA stability in wastewater.

Main Methods:

• Tested 14 virus concentration methods (centrifugation, filtration, PEG precipitation, ultrafiltration) and 4 RNA extraction methods.
• Used Bovine Respiratory Syncytial Virus (BRSV) as a process control for all 50 combined protocols.
• Analyzed SARS-CoV-2 RNA in wastewater influent samples using optimized methods and various RT-qPCR assays.

Main Results:

• The combination of centrifugation, ultrafiltration (Amicon-UF-15; 100 kDa cut-off), and Zymo Quick-RNA extraction provided the highest BRSV recovery (64.7%).
• BRSV RNA exhibited a first-order decay rate (k = 0.04/h) during prolonged storage at -20°C.
• SARS-CoV-2 RNA was detected in 81% of wastewater samples, with concentrations ranging from 1.10 × 10⁴ to 2.38 × 10⁶ gene copies/L.
• The US CDC N1 assay demonstrated the highest sensitivity among tested RT-qPCR assays.

Conclusions:

• Ultrafiltration coupled with Zymo Quick-RNA extraction is a highly effective method for SARS-CoV-2 concentration and recovery from wastewater.
• Optimized protocols enhance the reliability of wastewater-based epidemiology for SARS-CoV-2 surveillance.
• Understanding virus RNA stability during storage is critical for accurate wastewater monitoring.