https://doi.org/10.1016/j.mex.2024.102645

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. (2024) Solid-liquid distribution of SARS-CoV-2 in primary effluent of a wastewater treatment plant, MethodsX, Volume 12, 2024, 102645, ISSN 2215-0161

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.