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EVALUATION OF SELECTED DNA-BASED TECHNOLOGY IN IMPAIRED WATERSHEDS IMPACTED BY FECAL CONTAMINATION FROM DIVERSE SOURCES
Citation:
MOLINA, M. EVALUATION OF SELECTED DNA-BASED TECHNOLOGY IN IMPAIRED WATERSHEDS IMPACTED BY FECAL CONTAMINATION FROM DIVERSE SOURCES. U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-07/123 (NTIS PB2008-107359), 2007.
Impact/Purpose:
The objective of the proposed study is to evaluate and apply fast and reproducible DNA-based technology that can detect and track fecal contamination back to its source in complex environmental matrices, including recreational and drinking water resources.
Description:
Fecal pollution of surface waters is a top reason for impairment, as listed by the U.S. Environmental Protection Agency’s report on the quality of the Nation's waters. To be able to develop and implement TMDLs in impaired aquatic resources, it is imperative to determine the sources of the contamination. One tool used to determine the sources of bacterial fecal contamination is to apply a microbial source tracking approach to the system of interest. Microbial source tracking (MST) approaches are based on the assumption that specific strains of bacteria, genetic fingerprints, or DNA-based markers are associated with specific host species. Because accurate source identification of fecal contamination is essential in MST, more sensitive, selective and reliable molecular markers are required. The two types of genotypic methods that have been applied widely in a variety of environments can be classified as library-independent (LI) and library-dependent (LD). For both types of methods, the temporal and spatial stability of selected genotypes are aspects that need to be evaluated and are often times missing when applying MST to environmental samples. LD-MST methods require the development of large databases comprised of source specific isolates. Once a source specific fingerprint has been identified, the temporal and spatial variability of the particular genotype still needs to be validated. LI-MST is based on the application of culture-independent methods such as amplification of DNA from environmental samples using 16S rDNA markers in combination with polymerase chain reaction (PCR). However, cross-reactivity of some of the 16S rDNA markers used in field studies has prompted the development of alternative PCR assays using metagenomic markers specific for bovine feces. In this study we report on the comparison of selected LD and LI methodologies, their usability as rapid reliable methods for developing and applying markers to various environmental scenarios, and the stability of these markers under spatial and temporal considerations. From our results we concluded that library production is highly time and resource consuming. Its application is probably appropriate in very specific scenarios where discrimination among few selective sources is necessary. In contrast, application of DNA, PCR-based markers produced fairly rapid results and has the capability to screen multiple scenarios in a short period of time. Once stability and cross-amplification aspects have been addressed, it can be a highly efficacious approach to determine sources of contamination in a variety of scenarios.