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

Area of Science:

• Environmental microbiology
• Molecular epidemiology
• Public health surveillance

Background:

• Wastewater-based surveillance (WBS) tracks pathogens and predicts disease prevalence.
• SARS-CoV-2 WBS commonly uses nucleocapsid (N1, N2) and envelope (E) gene targets.
• Variability in results based on gene target choice requires investigation.

Purpose of the Study:

• To investigate variations between SARS-CoV-2 N2 and E gene targets in multiplex RT-ddPCR assays.
• To understand the reasons for observed differences in gene target performance during COVID-19 wastewater monitoring.

Main Methods:

• Analyzed over 700 raw wastewater samples from December 2021 to June 2022.
• Utilized a triplex RT-ddPCR assay with N2, E genes, and Murine Hepatitis Virus (MHV) as a process control.
• Conducted water quality tests (TSS, COD, pH, turbidity, NH3-N) and multiple regression analysis.

Main Results:

• The E gene concentration exceeded the N2 gene concentration by over one order of magnitude in more than 10% of samples.
• Higher TSS and COD levels negatively impacted viral recovery, indicating potential PCR inhibition.
• No clear relationship was identified between water quality variables or MHV recovery and gene concentration variance.

Conclusions:

• Primer selection significantly influences SARS-CoV-2 quantification in wastewater.
• Methodological standardization is crucial for reliable large-scale wastewater-based surveillance.
• Further research is needed to optimize gene targets for accurate WBS.