Exploration on wastewater-based epidemiology of SARS-CoV-2: Mimic relative quantification with endogenous biomarkers as internal reference

Wastewater-based epidemiology has become a powerful surveillance tool for monitoring the pandemic of COVID-19. Although it is promising to quantitatively correlate the SARS-CoV-2 RNA concentration in wastewater with the incidence of community infection, there is still no consensus on whether the viral nucleic acid concentration in sewage should be normalized against the abundance of endogenous biomarkers and which biomarker should be used as a reference for the normalization. Here, several candidate endogenous reference biomarkers for normalization of SARS-CoV-2 signal in municipal sewage were evaluated. The human fecal indicator virus (crAssphage) is a promising candidate of endogenous reference biomarker for data normalization of both DNA and RNA viruses for its intrinsic viral nature and high and stable content in sewage. Without constructing standard curves, the relative quantification of sewage viral nucleic acid against the abundance of the reference biomarker can be used to correlate with community COVID-19 incidence, which was proved via mimic experiments by spiking pseudovirus of different concentrations in sewage samples. Dilution of pseudovirus-seeded wastewater did not affect the relative abundance of viral nucleic acid, demonstrating that relative quantification can overcome the sewage dilution effects caused by the greywater input, precipitation and/or groundwater infiltration. The process of concentration, recovery and detection of the endogenous biomarker was consistent with that of SARS-CoV-2 RNA. Thus, it is necessary to co-quantify the endogenous biomarker because it can be not only an internal reference for data normalization, but also a process control.