Vetmax xeno internal positive control

To monitor the recovery performance of the SARS-CoV-2 RNA biomarker detection workflow, the bacteriophage Phi 6 was spiked into each sample (500 PFU.mL⁻¹ final concentration) prior to sample concentration. PCR inhibition was monitored in the aqueous and particulate phases using a synthetic internal positive control (IPC) (VetMAX™ Xeno™ Internal Positive Control, A29762 and VetMAX™ Xeno™ Internal Positive Control - VIC™ Assay, A29767; ThermoFisher Scientific), which was added to the RT-qPCR mix following the instructions of the manufacturer. To determine PCR inhibition, IPC DNA was added to positive control wells (SARS-CoV-2 RNA, EVAg-GLOBAL material) and the mean Cq value ± standard deviation (n = 18) was used as reference point to determine if inhibition occurred in wastewater samples. The latter were considered to have PCR inhibition if the Cq values of the IPC exceeded the reference point by >2 Cq values (Ahmed et al., 2020d ). Each RT-qPCR reaction was run in triplicates, both for standards and microcosm samples. To minimize the risk of RT-qPCR contamination, RNA extraction and RT-qPCR analysis were performed in separate laboratories. Extraction blanks were added to each RNA extraction run and no template controls were added to each RT-qPCR plate.