Chip-Based Digital PCR Approach for Improved SARS-CoV-2 Quantification for Endemic Wastewater Surveillance

The COVID-19 pandemic has revealed the importance of wastewater-based epidemiology (WBE) due to its advantages over traditional surveillance systems. However, low viral prevalence poses a significant challenge for reliable detection of SARS-CoV-2 in endemic wastewater settings. This study aimed to improve detection sensitivity of SARS-CoV-2 by applying the chip-based digital PCR (dPCR) approach targeting the CDC N1 and N2 genes to wastewater samples processed with a combined magnetic bead concentration method during a low-prevalence period in South Korea. Here, the evaluation results using dPCR combined with magnetic bead concentration showed higher viral loads and lower variability compared to the commonly used electronegative filtration method. Comparative analysis of the dual-target N1 + N2 and E-Sarbeco assays revealed that the N1 + N2 assay yielded significantly higher concentrations (4.73 ± 0.19 log₁₀ GC/L) than the E gene assay (4.27 ± 0.25 log₁₀ GC/L) in dPCR analysis (p < 0.01). PMMoV normalization increased the Pearson correlation coefficient between SARS-CoV-2 concentrations and clinical case data from R = 0.1 to R = 0.31; however, this was not statistically significant. These findings contribute to enhancing detection sensitivity for SARS-CoV-2 wastewater surveillance in endemic settings.