Comparison of PMMoV RNA, crAssphage DNA and human mitochondrial DNA/RNA as normalization markers in wastewater-based epidemiology

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Publication: Gallagher et al., Science of the Total Environment (2026)

Overview

This study compared three of the most widely used wastewater normalization biomarkers—Pepper Mild Mottle Virus (PMMoV) RNA, crAssphage DNA, and human mitochondrial cytochrome B (MT-CYB) DNA/RNA—to determine their suitability for wastewater-based epidemiology (WBE). Using a newly developed four-target multiplex RT-qPCR assay, researchers analyzed 922 wastewater samples from 29 wastewater treatment plants covering approximately 49% of Denmark's population. The study evaluated each marker's ability to normalize for wastewater dilution, estimate contributing population size, and improve SARS-CoV-2 surveillance. Samples were concentrated using Nanotrap® Microbiome A Particles prior to nucleic acid extraction and analysis.

Key Findings

Robust multiplex assay for wastewater normalization

• Researchers developed a single multiplex RT-qPCR assay capable of simultaneously quantifying PMMoV RNA, two crAssphage DNA targets, and MT-CYB DNA/RNA with high sensitivity and PCR efficiencies near 100%.

• All biomarkers were abundant in wastewater and demonstrated strong analytical performance across the national surveillance program.

All biomarkers effectively normalized wastewater dilution

• PMMoV, crAssphage, and MT-CYB performed similarly for correcting wastewater dilution, reducing variability compared with raw pathogen concentrations.

• Marker performance was influenced more by wastewater treatment plant-specific characteristics and extreme flow events than by the biomarker itself.

MT-CYB demonstrated the greatest overall stability

• Among the three markers, MT-CYB DNA/RNA showed the highest temporal and spatial stability, exhibiting minimal seasonal variation and the most consistent estimates of contributing population size.

• PMMoV displayed the lowest day-to-day variability but showed a pronounced seasonal increase during summer, while crAssphage exhibited modest seasonal variation.

Improved pathogen surveillance

• Normalizing SARS-CoV-2 wastewater measurements with any of the three biomarkers reduced variability compared with both raw viral concentrations and conventional flow- and population-based normalization.

• Biomarker normalization produced more stable estimates of SARS-CoV-2 trends, supporting improved interpretation of national wastewater surveillance data.

Implications for Nanotrap Technology

This study demonstrates that Nanotrap Microbiome A Particles provide a reliable sample concentration platform for national-scale wastewater surveillance, supporting sensitive and reproducible quantification of multiple normalization biomarkers across diverse wastewater systems. By enabling consistent recovery of human-associated nucleic acids prior to multiplex RT-qPCR, Nanotrap technology supports robust wastewater normalization workflows that improve pathogen trend analysis and population-level epidemiology.

Conclusion

The authors conclude that all three normalization biomarkers substantially improve wastewater pathogen surveillance, with MT-CYB DNA/RNA providing the most robust overall performance due to its superior temporal and spatial stability. Combined with Nanotrap Microbiome A Particle concentration, these biomarkers provide a reliable framework for improving normalization, reducing analytical variability, and strengthening wastewater-based public health surveillance.

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