Validation of methods for enriching and detecting SARS-CoV-2 RNA in wastewater

Abstract

Monitoring virus concentrations in wastewater is crucial for tracking community viral spread, yet reliable virus enrichment and detection methods are lacking. We compared four preconcentration-detection methods for tracing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater: ultrafiltration and covalent affinity resin separation, paired with either reverse transcription-quantitative polymerase chain reaction (PCR) or reverse transcription-digital PCR. Tests were conducted on raw sewage from a campus dormitory septic tank in May 2023. Results show that the concentrations of coronavirus RNA in wastewater enriched by ultrafiltration were higher than those enriched by covalent affinity resin separation, regardless of the detection methods used. Digital PCR exhibited detection rates of 45.4% using covalent affinity resin separation, and 77.3% using ultrafiltration. These detection rates were 36.4% and 72.7% higher than those using quantitative PCR. A correlation coefficient of 0.70 between concentrations measured by quantitative and digital PCR was only observed for samples enriched by covalent affinity resin separation.