Measuring the efficiency of a solids-based wastewater extraction using BCoV and validation of a BCoV and PMMoV RT-qPCR duplex assay.

Bovine coronavirus (BCoV) is commonly used to evaluate the efficiency of nucleic acid extraction protocols used in wastewater-based surveillance (WBS). Here we outline a method for measuring the efficiency of a solids-based extraction protocol using RT-qPCR detections of BCoV spiked into wastewater samples. Validation steps included: determination of a suitable quantification method for BCoV starting material, measures of endogenous BCoV in wastewater, assessment of the effects of BCoV incubation times on wastewater extraction efficiencies, and implementation of a BCoV and Pepper Mild Mottle Virus (PMMoV) RT-qPCR duplex assay. Results show that an accurate baseline measure of BCoV stock concentrations is best achieved using heat lysis extraction compared to an automated method. We also show that endogenous BCoV can be present in wastewater, albeit at low viral loads. Additionally, a BCoV incubation time of 16 h in wastewater resulted in the highest percent recovery. Lastly, the use of the BCoV and PMMoV RT-qPCR duplex assay demonstrates comparable performance to their singleplex counterparts. • A method for measuring the extraction efficiency of a solids-based wastewater extraction process using BCoV. • Insights into the in situ prevalence of BCoV in wastewater samples. • Assessment of a multiplex assay for simultaneous detection of BCoV and PMMoV.