Chicken- and duck-associated Bacteroides–Prevotella genetic markers for detecting fecal contamination in environmental water

Abstract

Bacteroides-Prevotella group is one of the most promising targets for detecting fecal contamination in water environments, principally due to its host-specific distributions and high concentrations in feces of warm-blooded animals. We developed real-time PCR assays for quantifying chicken/duck-, chicken-, and duck-associated Bacteroides-Prevotella 16S rRNA genetic markers (Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac). A reference collection of DNA extracts from 143 individual fecal samples and wastewater treatment plant influent was tested by the newly established markers. The quantification limits of Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac markers in environmental water were 54, 57, and 12 copies/reaction, respectively. It was possible to detect possible fecal contaminations from wild ducks in environmental water with the constructed genetic marker assays, even though the density of total coliforms in the identical water samples was below the detection limit. Chicken/Duck-Bac marker was amplified from feces of wild duck and chicken with the positive ratio of 96 and 61%, respectively, and no cross-reaction was observed for the other animal feces. Chicken-Bac marker was detected from 70% of chicken feces, while detected from 39% of cow feces, 8.3% of pig feces, and 12% of swan feces. Duck-Bac marker was detected from 85% of wild duck feces and cross-reacted with 31% of cow feces. These levels of detection specificity are common in avian-associated genetic markers previously proposed, which implies that there is a practical limitation in the independent application of avian-associated Bacteroides-Prevotella 16S rRNA genetic markers and a combination with other fecal contamination markers is preferable for detecting fecal contamination in water environments.