Detailed project information for Study Plan Number 01127

Branch :	Fish Health Branch
Study Plan Number :	01127
Study Title :	Method verification of the detection of mitochondrial cytochrome b sequences of DNA specific to various animal hosts in a set of challenge water samples
Starting Date :	07/15/2006
Completion Date :	09/30/2007
Principal Investigator(s) :	Schill, Bane (William)
Primary PI :	Schill, Bane (William)
Telephone Number :	(304) 724-4438
Email Address :	wschill@usgs.gov
SIS Number :
Primary Program Element :
Second Program Element :
Status :	Active
Abstract :	BACKGROUND Stoeckel and others (2004) compared the ability of seven library-dependent microbial source tracking protocols to associate a challenge set of Escherichia coli bacteria isolates to correct animal source. Correct rates of classification of animal sources of these challenge bacteria isolates did not meet literature expectations. Additional method testing with larger known-source libraries did not improve correct rates of classification of animal sources. A study (report in preparation) to determine animal sources of contamination in four tributaries to the New River demonstrated the ability of several library-independent sourcing protocols (most notably a host DNA method using mitochondrial DNA sequences) to detect the correct animal source of scat in blinded quality-control samples. The host DNA method was able to correctly detect human DNA in a blinded water sample containing human scat and to correctly detect deer and goose DNA in a blinded water sample containing deer and goose scat. The method was still able to detect the DNA even after several dilutions of the original sample. OBJECTIVES The objectives of this study are as follows: 1. To test the ability of the host DNA methodology to identify the correct source of scat in blinded quality-control samples containing one of nine scat samples from either human, dog, deer, goose, cow, pig, horse, chicken, or sheep. (Task 1) 2. To test the ability of the host DNA methodology to identify the correct proportions of blinded mixtures of 1-3 different scat samples per water sample. (Task 2) 3. To determine the general detection limits of the host DNA methodology by trying to detect the DNA in various known dilutions of water samples containing individual scat. Bacteria concentrations will be determined for each dilution to show how effectively each animal DNA can be detected relative to the concentration of indicator bacteria in the sample. (Task 3) HYPHOTHESIS TO BE TESTED A study (report in preparation) to determine animal sources of contamination in four tributaries to the New River (WV) demonstrated the ability of a newly developed molecular method that targets human and animal mitochondrial DNA sequences directly to detect the correct source of scat in blinded quality-control samples. This new study will further test the ability of the method to determine the source(s) and composition of fecal contamination in water and will test the sensitivity of the method relative the enumeration of common used microbial indicators including total coliform bacteria, Escherichia coli, and Enterococcus.
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