MP13 Aquatic Ecotoxicology
Monday, 14 November 2005: 8:00 AM - 6:30 PM in Exhibit Hall

(AND-1117-853412) The use of terminal restriction fragment length polymorphism (T-RFLP) in fecal bacterial source tracking.

Anderson, J¹, Farmer, J², Johnson, T², Bailey, F¹, ¹ Middle Tennessee State University, Murfreesboro, TN, USA² Tennessee State University, Nashville, TN, USA

ABSTRACT- Fecal pollution of environmental waters is a serious health problem that cannot be remedied until sources are identified. This study examines the hypothesis that terminal restriction fragment length polymorphism (T-RFLP) can be used in fecal source tracking. T-RFLP is a PCR-based method used in determining the genetic diversity within communities of organisms. Generally, each group within the community contributes at least one unique terminal DNA fragment. The T-RFLP procedure has been successful in profiling certain bacterial populations within fecal communities. However, in terms of fecal source tracking, most molecular methods employ the use of fecal bacterial libraries. We are exploring the use of T-RFLP as a non-library dependent method for aquatic fecal source tracking. A non-library dependent method will provide a quicker and less expensive way of determining the source of fecal contamination and may be less region-specific. Fecal bacterial DNA has been extracted from human, bovine, and sewage samples. Labeled and unlabeled 8F primers, labeled 516F, and unlabeled 1541R primers were used for PCR amplification. The samples were cut with the restriction enzymes Hae III and Hha I. After restriction cutting, differences were found among the human, bovine and sewage groups. T-RFLP analysis of the samples is now in progress. We expect that the terminal fragments of the three samples will be unique enough to allow differentiatiation between the source groups, thus making T-RFLP a tool for use in fecal source tracking.

Key words: bacterial source tracking, t-RFLP, fecal source tracking