Solid-liquid distribution of SARS-CoV-2 in primary effluent of a wastewater treatment plant

• Published in: MethodsX 2024-06, Vol.12, p.102645-102645, Article 102645
• Main Authors: Oloye, Femi F., Xie, Yuwei, Asadi, Mohsen, Challis, Jonathan K., Osunla, Charles A., Xia, Pu, Cantin, Jenna, Femi-Oloye, Oluwabunmi P., Brinkmann, Markus, McPhedran, Kerry N., Sadowski, Mike, Pandey, Sudhir, Jones, Paul D., Mangat, Chand, Servos, Mark R., Giesy, John P.
• Format: Article
• Language: English
• Subjects: COVID-19; Environmental RNA; Method development for SARS-CoV-2 RNA partitioning in wastewater; PCR inhibitors; Surveillance; Wastewater-based epidemiology
• ISSN: 2215-0161; 2215-0161
• DOI: 10.1016/j.mex.2024.102645

Distributions of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and fecal viral biomarkers between solid and liquid phases of wastewater are largely unknown. Herein, distributions of SARS-CoV-2, Pepper Mild Mottle Virus (PMMoV), and F-RNA bacteriophage group II (FRNAPH-II) were determined by viral RNA RT-qPCR. Comparison of viral recovery using three conventional fractionation methods included membrane filtration, a combination of mid-speed centrifugation and membrane filtration, and high-speed centrifugation. SARS-CoV-2 partitioned to the solids fraction in greater abundance compared to liquid fractions in a combination of mid-speed centrifugation and membrane filtration and high-speed centrifugation, but not in membrane filtration method in a particular assay, while fecal biomarkers (PMMoV and FRNAPH-II) exhibited the reciprocal relationship. The wastewater fractionation method had minimal effects on the solids-liquids distribution for all viral and phage markers tested; however, viral RNA load was significantly greater in solid–liquid fractions viral RNA loads compared with the than whole-wastewater PEG precipitation. A RNeasy PowerWater Kit with PCR inhibitor removal resulted in greater viral RNA loads and lesser PCR inhibition compared to a QIAamp Viral RNA Mini Kit without PCR inhibitor removal. These results support the development of improved methods and interpretation of WBE of SARS-CoV-2. •Distribution of SARS-CoV-2 to liquid and solid portions was addressed.•Addressing PCR inhibition is important in wastewater-based epidemiology.•Fraction methods have minimal effect. [Display omitted]