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Fecal Source Tracking Using Human and Animal DNA

Fecal Source Tracking Using Human and Animal DNA. Bane Schill- USGS Leetown Science Center. U.S. Department of the Interior U.S. Geological Survey. Poop!. Fecal Source Tracking Using Human and Animal DNA. Bane Schill- USGS Leetown Science Center. U.S. Department of the Interior

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Fecal Source Tracking Using Human and Animal DNA

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  1. Fecal Source Tracking Using Human and Animal DNA Bane Schill- USGS Leetown Science Center U.S. Department of the Interior U.S. Geological Survey

  2. Poop! Fecal Source Tracking Using Human and Animal DNA Bane Schill- USGS Leetown Science Center U.S. Department of the Interior U.S. Geological Survey

  3. Overview- • History • Methodologies • Recent Study • Future Directions

  4. Water Availabililty

  5. E. Coli Enterococcus Bacteroides spp. Clostridium spp Bifidobacterium Pathogenic bacteria Pathogenic virus

  6. Yuck!!!

  7. Who Done It???

  8. Methodologies • Source tracking has been attempted using- • Biological Methods • Speciation of indicator bacteria • Genotyping of indicator bacteria • Identification of host-specific virus • Biochemical Methods • Fecal sterols • Stable isotope ratios • Brightening agents • Pharmaceuticals

  9. Methodologies • Most methods are not generally applicable for • one reason or another- • Biological Methods • Speciation of indicator bacteria (no fidelity to host) • Genotyping of indicator bacteria (specific strains low in frequency, antibiotic resistance variable, library-based methods expensive) • Identification of host-specific virus (Not available for all species, expensive) • Biochemical Methods • Not specific

  10. What about direct detection of human/animal DNA? • Pros- • Absolutely specific • Large, publicly available database (GenBank) • Methods for amplification and quantitative detection available • Cons- • Amounts found in receiving waters unknown • Halflife in water unknown • Methods for isolation from large volumes lacking

  11. The Cell Nucleus Mitochondria (150 – 2,600 per cell) mtDNA (about 16,500 bp)

  12. Who Done It??? Dissolved DNA Free Mitochondria Epithelial Cells in Feces Degraded Degraded Degraded Bound to Sediment

  13. Standard overnight culture • Fecal coliforms • E. Coli • Enterococcus • Polymerase Chain Reaction • Bifidobacterium • Bacteroides • Virulence determinants Bacteria Virus “Naked” DNA Bacteria on filter Sample Precipitation/Ultrafiltration Virus “Naked” DNA • Polymerase Chain Reaction • Enterovirus • “Naked” DNA

  14. Quantitative PCR Probe Primer Primer Polymerase Fluorescent Reporter Quencher

  15. Human mtDNA 10-fold Dilution Series

  16. Then a miracle happens (qPCR) that allows us to measure minute amounts of DNA

  17. At first only human DNA, then human and cow, then— Time Passes. . . . . .

  18. Horse1 Horse2 Dog Human Cow Sheep Deer Chicken Goose Pig

  19. Open File Reports-

  20. Challenge of Method With Blinded Samples • Preparation of Twenty Blinded Fecal Suspensions- • One gram feces was homogenized in 24ml dH2O • Strained through 40 micron nylon mesh • Suspension (0.25 ml) was added to 200 ml PBS • Final feces concentration was 5 mg/100 ml • Isolation and Concentration of Fecal DNA from Suspensions- • Suspension (100 ml) was passed through a 0.22 micron filter • Calcium, magnesium, sodium chloride, and EtOH were added to the filtrate • The divalent cation/DNA complexes were recovered by centrifugation • The complexes were broken with EDTA and DNA was recovered by ultrafiltration • Concentrated DNA was combined with material retained by filters, and total DNA was purified using a commercial kit.

  21. Journal Publication- Schill, W. B., and M. V. Mathes. 2008. Real-Time PCR Detection and Quantification of Nine Potential Sources of Fecal Contamination by Analysis of Mitochondrial Cytochrome b Targets. Environ. Sci. Technol. 42:5229-5234.

  22. Summary of Study Results- Significant findings are summarized in the following slides.

  23. Twenty blinded fecal suspension challenge samples were analyzed and identified with high specificity (0.994) and sensitivity (0.850). Sample number Sample composition Identification Sensitivity Specificity 1 white-tailed deer white-tailed deer 1.000 1.000 2 Canada goose Canada goose 1.000 1.000 3 white-tailed deer white-tailed deer 1.000 1.000 4 dog below detectiona0.000 1.000 5 dog dog > chickenb1.000 0.875 6 Canada goose Canada goose 1.000 1.000 7 human human 1.000 1.000 8 blank blank 1.000 1.000 9 human human 1.000 1.000 10 human human 1.000 1.000 11 horse horse 1.000 1.000 12 blank blank 1.000 1.000 13 sheep sheep 1.000 1.000 14 cow (beef) below detectionc0.000 1.000 15 cow (dairy) cow 1.000 1.000 16 chicken below detection 0.000 1.000 17 horse horse 1.000 1.000 18 sheep sheep 1.000 1.000 19 chicken chicken 1.000 1.000 20 pig pig 1.000 1.000 aSignal just below established threshold of detection (see text). bDog signal dominant with chicken signal just above established threshold of detection. cA second subsample was extracted and reanalyzed with the same outcome.

  24. Future Directions- • Future efforts include- • Expansion of the species assays to include turkey. • Testing whole-genome amplification and gene capture methods to increase sensitivity. • Testing methods for field preservation of samples and ways to streamline sample preparation. • Standardization of quality assurance/ quality control procedures. • Development of multiplexed assays.

  25. Melting Curve Analysis-

  26. Luminex-

  27. Target specific probe segment • Quantification of 100 • Analytes Simultaneously • DNA Sequences • RNA Sequences • Proteins • Others possible

  28. Thanks! Questions and Comments?

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