Q-260. Novel PCR Assays Based on Bacteroidales 16S Ribosomal RNA Genes for Faecal Source Identification in Water

S. Dorai-Raj, E. Colleran;
Natl. Univ. of Ireland, Galway, IRELAND.

Background: Detection of faecal contamination of water has traditionally been based on the enumeration of faecal coliforms, E. coli and faecal enterococci. These methods do not permit differentiation between human and non-human sources of faecal contamination, information which would be valuable in targeting of water source protection measures. The aim of this study was to develop a molecular assay based on the 16S rRNA gene of the order Bacteroidales for specific identification of ruminant faeces in water. Methods: Ruminant-specific 16S rRNA gene sequences were identified using terminal fragment length polymorphism (TRFLP) analysis and sequencing of cloned fragments. Two primer pairs based on novel sequences identified in this study, combined with a previously published primer, were subject to evaluation on DNA extracted from ruminant, human, avian, canine, equine and porcine faecal samples. Limits of detection were defined with serial tenfold dilutions of (a) water contaminated with a defined quantity of faeces and (b) target Bacteroidales 16S rDNA fragments. The ruminant-specific PCR assays were also tested on naturally contaminated samples from three rural drinking water supplies where land use patterns indicates ruminant faeces as the main source of faecal pollution. Results: One of the ruminant-specific PCR assays amplified DNA from 100% of the 74 adult ruminant faecal samples tested, 0% of the 59 human sewage/faecal samples and 9% of the 44 non-ruminant animal faecal samples. The other ruminant-specific PCR assay amplified DNA from 90.5% of the adult ruminant faecal samples tested and 0% of the human and non-ruminant animal sewage/faecal samples. The limit of detection was from 7.3 × 10-5 to 7.3 × 10-6 g (dry weight) of faeces/litre and from 1 ×102 to 1 ×104 copies of 16S rDNA. The two PCR assays amplified the ruminant-specific Bacteroidales 16S rDNA fragments from many of the naturally contaminated rural water samples. The most sensitive of the assays was positive in water with circa 50 or greater E. coli per 100 ml. Conclusions: The use of these two assays in combination shows promise for faecal source identification studies and they merit further field testing and specificity evaluation.