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CULTURE-INDEPENDENT MOLECULAR METHODS FOR FECAL SOURCE IDENTIFICATION
Citation:
FIELD, K. G., S. WUERTZ, AND O. C. SHANKS. CULTURE-INDEPENDENT MOLECULAR METHODS FOR FECAL SOURCE IDENTIFICATION. Presented at Water Environment Research Foundation, Microbial Source Tracking Workshop, San Antonio, TX, February 17, 2005.
Impact/Purpose:
To inform the public
Description:
Fecal contamination is widespread in the waterways of the United States. Both to correct the problem, and to estimate public health risk, it is necessary to identify the source of the contamination. Several culture-independent molecular methods for fecal source identification have been proposed. These methods are fast (less than 24 hours) and inexpensive, don't require culturing, and don't require site-specific libraries. The methods are based on extracting DNA from water samples and amplifying diagnostic gene sequences. Proposed targets for culture-independent molecular methods include virulence genes from E. coli and enterococci, cultivated species of Bifidobacterium and host-specific groups of Bacteroidales fecal bacteria. Issues include sensitivity, specificity, and stability. Sensitivity is affected by DNA extraction efficiency and PCR inhibition. The addition of a known quantity of surrogate cells or markers to samples (for example, the phage PP7 for viral assays, and an appropriate bacterial species for bacterial assays) allows an estimation of DNA extraction efficiency and PCR inhibition. Because pathogens are rare and sporadically distributed, growth may be necessary to amplify virulence genes signals enough to detect them. Thus the virulence-gene-based assays may not be truly culture-independent. Fecal anaerobes comprise a higher proportion of fecal bacteria than do indicator bacteria, making them an appropriate target for detection without growth; however, little is known about their survival after release. Thus far, culture-independent molecular methods offer the fastest and least inexpensive method of documenting human fecal pollution. More markers for animal species are needed. The approach could be improved by the development of multiple, redundant markers for humans and other species of interest. In addition, more information is needed about the survival of markers in relation to each other and to pathogens. Finally, epidemiological studies are needed to establish human health risks posed by fecal pollution from different animal sources