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Parasites

Quantitative Real-Time PCR as a tool for the quantification and characterization of microorganisms in caves and karst aquifers: Phytoplankton , lampenflora, bacterial communities, and fecal source tracking Rick Fowler Western Kentucky University Bowling Green, Kentucky USA.

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Parasites

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  1. Quantitative Real-Time PCR as a tool for the quantification and characterization of microorganisms in caves and karst aquifers: Phytoplankton, lampenflora, bacterial communities, and fecal source tracking Rick Fowler Western Kentucky University Bowling Green, Kentucky USA

  2. Quantitative Real-Time PCR as a tool for the quantification and characterization of microorganisms in caves and karst aquifers: Phytoplankton, lampenflora, bacterial communities, and fecal source tracking Rick Fowler Western Kentucky University Bowling Green, Kentucky USA

  3. Fungi Parasites Bacteria Protists Algae Zooplankton Organisms suitable for DNA analysis

  4. Fungi Parasites Bacteria Protists Algae Zooplankton Organisms suitable for DNA analysis

  5. Polymerase Chain Reaction (PCR) • Many copies of specific DNA sequence • Quantitative Real-Time PCR • (qRT-PCR) • Many copies of specific DNA sequence • Fluorescent dyes • Quantification and melting point analysis

  6. Combination of compatible Dyes: • SYBR Green • HEX • NED • ROX, VIC, Cy3, EB, BHQ, etc.

  7. 0.08ng qRT-PCR data to quantify specific DNA sequences 0.4ng 0.000064ng 0.00032ng 0.0016ng

  8. Algae and phytoplankton in surface streams and cave springs Lampenflora

  9. Green River main channel Turnhole Bend cave spring “Blue Hole”

  10. Chlorella algae is a major food source for endangered mussel species in surface streams Chlorella is also found in lampenflora near cave tourist trails

  11. vulgaris Cave DNA 1 kb Ladder 1 kb Ladder Cave DNA 1000 ml Chlorella Chlorella 1000 ml 1300 ml 1300 ml 100 ml 750 ml 250 ml 750 ml 250 ml 250 ml 500 ml 500 ml 100 ml DNA test is specific and sensitive for Chlorella Environmental DNA from karst surface stream Purified DNA from different aquatic organisms Chlamydomonas reinhardti vulgaris Chlorella vulgaris Decapods 1 kb Ladder Insects Cave DNA Bacteria Fungi DNA Extracts 1000 500 250 200 bp PCR Product 10,000 3000 1000 1000 500 PCR 200 500 Products 100

  12. Surface flow | Subsurface springs 100 10 1 0.1 0.01 0.001 0.0001 GRGR NRNR BIGR HRTH ERES BSBS DFDF BSBC Blank Chlorella DNA concentrations in Mammoth Cave National Park Chlorella DNA(ppt)

  13. Cave bacteria

  14. Quantification of bacterial DNA in cave sediments

  15. Artificial substrates were beads containing limestone 1 mm

  16. Beads were placed in dye-trace bags and recovered after one year in cave streams Limestone Ca13CO3

  17. Quantification of bacterial DNA on artificial substrates at five study sites

  18. Phylogenetic trees of cave bacteria Many cave bacterial 16SrDNA genes were sequenced

  19. Lipid Analysis of artificial substrates Different technique based on analysis of membrane lipids

  20. Microbial Source Tracking Identification of the origins of fecal pollution in karst aquifers

  21. Quantitative Real Time PCR Determination Bacteroides 16S rDNA concentration SYBR Green

  22. Melting point analysis Bacteroides 16S rDNA from different animals can be differentiated SYBR Green

  23. HEX-Bac32F primer HEX-Bac32F primer Bacteroides 16S rDNA Bacteroides 16S rDNA HEX HEX HEX HEX HEX HEX HEX HEX HEX HEX NED NED qRT-PCR NED-1492r primer NED-1492r primer Dual-labeled Bacteroides 32f-798r amplicons NED C T T C NED C C C NED NED T T T T NED NED Scheme for microbial source tracking SYBR Green Dual-labeled Bacteroides 32f-798r amplicons NED C T T C NED C

  24. Human Sewage Horse

  25. Acknowledgements Web site http://people.wku.edu/rick.fowler/mammoth

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