Fluorescence damping as primary interference mechanism of humic acids on qPCR quantification of SARS-CoV-2 in wastewater surveillance

Real-time polymerase chain reaction (qPCR) is a widely used technology for the detection and quantification of nucleic acids in wastewater surveillance. However, the presence of inhibiting and/or interfering substances in wastewater samples, in particular humic acids, can significantly impact the accuracy and reliability of qPCR results. In this study, we investigated the impact of humic acids on qPCR reactions and determined the relative importance of various inhibition/interference mechanisms through spiked experiments and modeling. Our results showed that higher concentrations of humic acids led to increasing threshold cycle (C_T) values, which however cannot be adequately described by the polymerase inhibition model and the DNA template complexation model. Further inspection showed that humic acids caused fluorescence damping of the FAM reporter dye, resulting in an overall decrease in fluorescence intensity. Modeling of the fluorescence damping effect showed that the C_T values of qPCR reactions can be corrected based on end-point fluorescence reduction. Similar observations and corrections were also achieved when SARS-CoV-2 cDNA was spiked with an actual wastewater sludge cDNA.