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DNA BASED PREDATOR STOMACH CONTENT
ANALYSIS FOR SINGLE AND MULTIPLE PREY
SPECIES USING QUANTITATIVE POLYMERASE
CHAIN REACTION (QPCR)
Gregg Schumer, Cramer Fish Sciences/Genidaqs
Scott Brandl, University California Davis
Melinda Baerwald, University California Davis
Brian Schreier, California Department of Water Resources
Veronica Wunderlich, California Department of Water
Resources
Scott Blankenship, Cramer Fish Sciences/Genidaqs
BRIEF OVERVIEW
OF POLYMERASE
CHAIN REACTION
(PCR)
DNA copies
PCR was first described in
1983 by Kerry Mullis and
awarded the Nobel Prize
in 1993
PCR “primers” are designed to
amplify targeted
sequences of DNA giving
PCR its inherent specificity
Cycle
1 cycle
Quantitative Real
Time Polymerase
Chain Reaction
(qPCR)
qPCR follows the principles of PCR
(primer selectivity); its key feature is
that with the addition of a labeled probe
the amplified DNA is detected as the
reaction proceeds in real time.
QPCR AS A MONITORING TOOL :
STOMACH CONTENT ANALYSIS
 Traditional Visual ID
 Molecular ID:
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Laborious
Time consuming
Expensive
Ineffective in many instances
Limited by the sample
qPCR
Rapid
Cost effective
Highly specific
High throughput
Not limited by sample type
All methods have been thoroughly
vetted(SNP genotyping, disease
detection, gene expression)
DNA Sequence
TTAATTCAACTACAAGAACCC
TAATGGCCAACCTTCGGAAA
Design and
validation of
species
specific
q-PCR assays
TTAATTCAACTACAAGAACCC
TAATGGCCAACCTTCGGAAA
1. Determine species
“barcode” CO1, CytB
2. Design species specific
primer probe sets
(qPCR assay design)
3. Validate primer probe
sets
4. Test samples presence/absence for
species of interest
General workflow for
stomach contents
analysis
Presence
absence of
target species
Lab Feeding Trial
Detection rate vs time
MSS eating DS
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
0
10
20
30
40
50
60
Genomic Variation Lab UC Davis
70
FIELD PILOT STUDY – APRIL 2010
658 dissected silversides
0
15
Shoals
Channel
Silversides positive for smelt
2011 EXPANDED FIELD STUDY
March - July
2011
Species
(Predators)
Number tested
for smelt DNA
Positive for
smelt DNA
Mississippi silverside
559
69
Striped bass
73
1
Pikeminnow
44
2
Sunfish
38
1
Largemouth bass
30
2
Gobies
25
1
Threadfin shad
21
1
Chinook salmon
16
1
Golden shiner
10
1
Tule perch
6
1
Exopalaemon shrimp
4
1
Three-spine
stickleback
1
1
If you can do this for a single prey species why not multiple prey species?
Multi Species/High Throughput
Fluidigm Dynamic Array Technology
Time
Labor
Reagents
Highlights
• Less time = Less cost
• Fewer reagents = less cost
• Dynamic format (species x samples)
• 48x48, 96x96, 24x192
Striped Bass Predation Study 2013-14
General workflow for
stomach contents
analysis
Presence
absence of
target species
Take home message
• When appropriate, qPCR is a viable technique for
detecting single species individually and multiple
species simultaneously.
• Once you have the DNA you can always go back and
re-analyze later
• Dynamic platform.
 Detection of different species from
different sample types
Acknowledgements
QUESTIONS?