Variabilities in N2 and E Gene Concentrations in a SARS-CoV-2 Wastewater Multiplex Assay

Wastewater can serve as both a source of pathogens that pose risks to human health and a valuable resource for tracking and predicting disease prevalence through wastewater-based surveillance (WBS). In WBS for SARS-CoV-2, both nucleocapsid-specific (N1 and N2) and the envelope (E) genes are common targets for primer design, but ambiguity remains regarding differences in results depending on the gene target chosen. This study investigated how and why two SARS-CoV-2 gene targets (N2 and E) varied when analyzed in a multiplex RT-ddPCR assay for a COVID-19 wastewater monitoring study. From December 2021 to June 2022, over 700 raw wastewater samples were collected from thirteen manholes in the University of California, Irvine sewer system. Murine hepatitis virus (MHV) was used as a matrix recovery and process control in the triplex RT-ddPCR assay. Water quality tests (TSS, COD, pH, turbidity and NH₃-N) were performed on all samples. Analyses showed that in over 10% of samples, the E gene concentration exceeded N2 by more than one order of magnitude. To evaluate matrix effects on amplification efficiency for N2 and E genes, multiple regression analysis was performed to explore whether water quality variables and MHV recovery efficiency could predict variance in gene concentrations, but no clear relationship was identified. However, viral recovery, as indicated by MHV recovery efficiency, was negatively impacted in samples with higher TSS and COD, suggesting PCR inhibition. These findings contribute to methodological standardization efforts in WBS and emphasize the importance of primer selection for large-scale monitoring.