Q-254. Significant Cross-Amplification of Fish Fecal DNA Using Quantitative Real-Time PCR Specific to the Human Bacteroides 16S rRNA Genetic Marker

L. Kabiri-Badr1, H. Ryu1, J. McLain2, C. Rock3, A. Alum1, M. Abbaszadegan1;
1Arizona State Univ., Tempe, AZ, 2USDA-ARS, USALARC, Maricopa, AZ, 3Univ. of Arizona, Maricopa, AZ.

Microbial source tracking is a valuable tool for identifying sources of fecal pollution in environmental samples. Bacteroides-specific molecular markers have been used to discriminate human fecal bacteria from other fecal sources, but some cross-reactivity of the human Bacteroides primers and probe with other fecal sources has been reported. We investigated the cross-reactivity of the primers and probe specific to human Bacteroides with known sources of fecal bacteria. A pond in Maricopa, Arizona was monitored for several months. The pond is filled with tertiary treated municipal effluent and is stocked for recreational fishing. High level of fecal bacteria was detected in the pond. Bacteroides-based source tracking using Q-PCR indicated that the majority of fecal contamination in the pond water was of human origin. The results were against expectations; therefore, the human-specific Bacteroides primers and probe were further investigated for their cross-reactivity against known sources of fecal contamination especially fish (tilapia, Tilapia spp.) feces. The source of the cross-reactivity was examined using direct cloning and DNA sequencing of PCR product from amplification of fecal DNA from humans and fish (tilapia and catfish (Siluiformes)) using the human Bacteroides-specific primers. Nucleotide sequences were determined for 15 clones from the human and fish PCR products, all products were the expected size (106 bp). All clones (human and fish) showed 100% homology to the Q-PCR probe sequence. The nucleotide sequences from all human clones were identical; however, mismatches were identified between the fish and human PCR products, indicating that the fish fecal bacteria were unique strains of Bacteroides, and that observed cross-reactivity was not the result of contamination of the fish fecal samples with human feces. These results may call into question the use of published Bacteroides-specific molecular markers in quantification of human fecal contamination in waters where fish may contribute to fecal inputs.