Work

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

Year: 2025

Type: article

Abstract: 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.

Source: Journal of Hazardous Materials

DOI: 10.1016/j.jhazmat.2025.138283

Authors: Min Ki Jeon, Tao Yan

Institution: University of Hawaiʻi at Mānoa

Language: English

Topic: SARS-CoV-2 detection and testing

Subfield: Infectious Diseases

Field: Medicine

Domain: Health Sciences

SDG: Clean water and sanitation

Open Access status: hybrid

Estimated APC paid: $4,340