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Development and application of an adsorption-direct extraction method for recovering nucleic acids of enteric viruses and host-specific Bacteroidales from groundwater.

In this study, the adsorption-elution method was modified to concentrate viral particles in water samples and investigate the contamination of groundwater with norovirus genogroup II (NoV GII), rotavirus A (RVA), and pepper mild mottle virus (PMMoV). The mean recovery rate of a murine norovirus strain, which was inoculated into groundwater samples collected from a deep well, was the highest (39%) when the viral RNA was directly extracted from the membrane instead of eluting the adsorbed viral particles. This adsorption-direct extraction method was applied to groundwater samples (20 L) collected from deep wells used for public drinking water supply (N = 22) and private wells (N = 9). RVA (85 copies/L) and NoV GII (35 copies/L) were detected in water samples from a deep well and a private well, respectively. PMMoV was detected in 95% and 89% of water samples from deep wells and private wells, respectively, at concentrations of up to 990 copies/L. The modified method could also be used to extract bacterial DNA from the membrane (recovery rate of inoculated Escherichia coli K-12 was 22%). The Bacteroidales genetic markers specific to ruminants (BacR) and pigs (Pig2Bac) were detected in samples from a deep well and private well, respectively. The modified virus concentration method has important implications for the management of the microbiological safety in groundwater supply. IMPORTANCE We investigated the presence of enteric viruses and bacterial genetic markers to determine fecal contamination in groundwater samples from deep wells used for public drinking water supply and private wells in Japan. Groundwater is often subjected to chlorination; malfunctions in chlorine treatment result in waterborne disease outbreaks. The modified method successfully concentrated both viruses and bacteria in 20 L groundwater samples. Norovirus GII, rotavirus A, pepper mild mottle virus, and Bacteroidales genetic markers specific to ruminants and pigs were detected. Frequent flooding caused by increased incidences of extreme rainfall events promotes the infiltration of surface runoff containing livestock wastes and untreated wastewater into wells, possibly increasing groundwater contamination risk. The practical and efficient method developed in this study will enable waterworks and the environmental health department of municipal/prefectural governments to monitor water quality. Additionally, the modified method will contribute to improving the microbiological safety of groundwater.