title: A high-throughput and quantitative hierarchical oligonucleotide primer extension (HOPE)-based approach to identify sources of faecal contamination in water bodies.
abstract: As faecal contamination of recreational and drinking water impairs the water quality and threatens public health, water bodies are routinely monitored for faecal coliforms to detect contamination. However, faecal coliforms are facultative anaerobes that survive and reproduce in ambient waters, and their presence does not depict the origin of contamination. Therefore, the use of Bacteroides-Prevotella 16S rRNA gene to perform faecal source tracking has been proposed and applied. Here, we demonstrate the use of a new molecular method termed hierarchical oligonucleotide primer extension (HOPE) to simultaneously detect human-associated Bacteroides spp. and three clusters of cow-, pig- and dog-specific uncultivated Bacteroidales. The method correctly identifies the origin of faecal contamination when tested against human, cow, pig and dog faeces (n = 17, 17, 16 and 13 respectively), and in waters contaminated with faeces of known origins. Subsequent tests with a total of 21 blind samples show that HOPE is able to accurately indicate single or multiple sources of faecal contamination originating from pigs, cows and humans in 81% of the blind samples. HOPE can further correctly detect and identify faecal contamination in five sampling sites located along a canal in southern Taiwan, and the results are validated against conventional faecal coliform tests and quantitative PCR. Overall, this study demonstrates HOPE as a quantitative and high-throughput method that can identify sources of faecal contamination.
authors.affiliation: Division of Environmental Science and Engineering, National University of Singapore, Singapore 117576.
authors.forename: Pei-Ying
authors.initials: PY
authors.lastname: Hong
authors.forename: Jer-Horng
authors.initials: JH
authors.lastname: Wu
authors.forename: Wen-Tso
authors.initials: WT
authors.lastname: Liu
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques; D001431:Bacteriological Techniques / Q000379:methods*; D041963:Bacteroidetes / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004285:Dogs; D005243:Feces / Q000382:microbiology*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D011634:Public Health / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013552:Swine; D013624:Taiwan; D014871:Water Microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S