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Project Number 2.5.16

Project Number 2.5.16. Project Title Development of a Rapid and Quantitative Genetic Based Asian Carp Detection Method. Wen-Tso Liu, University of Illinois at Urbana-Champaign Ryan Jackson, USGS (Illinois) Duane Chapman, USGS (Missouri) Jon Amberg / Mark Gaikowski, USGS (Wisconsin).

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Project Number 2.5.16

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  1. Project Number 2.5.16 Project Title Development of a Rapid and Quantitative Genetic Based Asian Carp Detection Method Wen-Tso Liu, University of Illinois at Urbana-Champaign Ryan Jackson, USGS (Illinois) Duane Chapman, USGS (Missouri) Jon Amberg / Mark Gaikowski, USGS (Wisconsin)

  2. Introduction and Objectives • Overall • To develop a rapid and quantitative genetic method for AC detection • Specific: • Identify Asian carp‐specific microbial populations in fish guts. • Develop Asian carp‐specific qPCR test. • Test Asian carp‐specific qPCR test with blind samples Bighead carp Black carp Grass carp Silver carp

  3. Hypothesis • Microbial source tracking of fecal contaminants • Asian carps and indigenous fish species have different gut microflora host-specific microbial populations. • Interspecies differences between Asian carps (due to diet/regional differences). • Host-specific microbial populations in fecal matters • Biomarker: 16S rRNA gene • 1014 cells/g fecal mass, ~200-1000 organisms Which poo(s)?

  4. Dog feces Sewage Surface water Human Total microbial populations McLellan et al. Environ Microbiol. 2010. 12(2):378 Dog Human Pig Cow Stress: 0.13 Environ Microbiol. 2009.11(7):1672-81. Hong et al. (2009) Environ Microbiol 11: 1672 Host-specific microbial populations

  5. HOPE:Pig-specific Bacteroidales FC:< 100 CFU/100ml Test 2 Environmental waters (Tainan, TW) HOPE: No host-specific biomarkers FC:< 100 CFU/100ml HOPE:Human-associated Bacteroides FC:7.4 x 103 CFU/100ml HOPE:Human-associated Bacteroides FC:1.3 x 104 CFU/100ml HOPE:Human-associated Bacteroides, Pig-specific Bacteroidales FC:4.7 x 106 CFU/100ml Hong et al. (2009) EM 11: 1672

  6. Current Knowledge: DNA sequencing technology • Next-generation sequencing technology Sanger Sequencing “Mega Project” -- the human genome has approximately 3.3 billion base-pairs (B bp) / ~500 DNA sequencing machines • Advantages • (per machine) • 5000 fold reduction in cost • 20 fold reduction in time • Reproducibility • Ease of use

  7. Current Knowledge PLoS One. 2010. 1;5(4):e9964. Proc Natl Acad Sci U S A. 20106;107(14):6477-81. Pyrosequencing allows for high-through put characterization of microbial communities from different environments

  8. Future Plans • Experiments: • Collection of fecal materials • Comparing microbial populations in fecal matters of different carps • Construction of 16S rRNA library for different carps • Identification of Asian carp-specific microbial populations • Design and validate Asian carp-specific qPCR test (quantitative DNA testing) • Interactions with other projects • Complement projects 2.5.10 and 2.5.12 • Do different gut sections (foregut, hindgut, midgut) harbor different microflora? • Can a better characterization of the gut microflora help us better understand Asian carp diet/metabolism? • Complement current surveillance methods, e.g. eDNA method • Increasing redundancy and sensitivity

  9. Technological or Other Hurdles • Representative fecal samples • Sample collection: • Replicates of different Asian carps feces • no contamination! • Comprehensive • Seasonal changes • Geographical locations • Diet • Identifying host-specific microbial populations? • Change with time, diet?

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