Fecal contamination of water is widespread in the United States, and of particular concern to the animal/poultry-agriculture community. Accurate, timely, and cost-effective methods of identifying fecal sources are critical to correcting water contamination problems. Current methods of Bacterial Source Tracking (BST) include a variety of both molecular and non-molecular techniques; however, the regulatory and source tracking communities are clearly moving towards non-library based methods that employ source-specific gene sequences. Such sequences can be the basis of proprietary DNA primers that can be subsequently used as probes, and when coupled with real-time PCR, fast and accurate results without the need for large and expensive known source libraries can be obtained. This project involves developing Gene Sequence Identification (GSI) methodology to determine the sources of E. coli and Enterococci in rural waters. This innovative approach will, if successful, provide significant improvements over current techniques for determining fecal contamination sources in water. OBJECTIVES: The overall goal of this project is to develop a new method for bacterial source tracking, Gene Sequence Identification (GSI). The specific objective is to identify gene sequences that are source specific through analysis of existing libraries and data developed through execution of this study. APPROACH: 1) Existing Pulsed-Field Gel Electrophoresis (PFGE) databases (E. coli and Enterococcus) will be mined to identify regions of the genome that are likely to contain source-specific gene segments. 2) Restriction Fragment Length Polymorphism (RFLP) analysis will be perfomed on identified regions of the genome to determine specific gene segments that are indicative of the host source. 3) Additional known-source bacteria will be analyzed using RFLPs of the PCR product from the 16S-23S Internal Transcribed Spacer (ITS) regions in both E. coli and Enterococcus 4) PCR-Ribotype libraries (E. coli and Enterococcus) will be mined to identify regions of the rRNA operons that are likely to contain source-specific gene segments
