Comparative Evaluation of DNA Extraction Workflows for Efficient Recovery of pBI143 from Wastewater

This study aimed to compare the performance of polyethylene glycol (PEG) precipitation, and Nanotrap ® Microbiome magnetic particle capture workflows for recovering novel fecal marker, pBI143 from 12 wastewater samples collected across six treatment plants in Maryland, USA. Quantitative PCR (qPCR) was used to quantify marker abundance. The Nanotrap workflow yielded significantly higher concentration of pBI143 compared to PEG precipitation workflow ( p < 0.05). The Nanotrap workflow used in the study utilized both magnetic nanoparticles A and B, rather than magnetic nanoparticle A alone, highlighting the necessity of optimization based on the intended targets for enhanced recovery. The extracted total nucleic acids by the Nanotrap workflow, were further analyzed to quantify other fecal markers, crAssphage, tomato brown rugose fruit virus (ToBRFV), and pepper mild mottle virus (PMMoV). No significant differences in the concentrations of pBI143, crAssphage, and ToBRFV ( p > 0.05) were observed, whereas the concentration of PMMoV was significantly lower than that of the three fecal markers ( p < 0.05). Based on the concentration alone, pBI143, ToBRFV, and crAssphage were found to be a better alternative to PMMoV as an endogenous fecal marker.