Bryce Hostetler (REU) , Melissa Hess (RET) , Laura Borecki , Dr

Optimization of qPCR Techniques to Determine Environmental DNA Transport in Stream Systems
Bryce Hostetler
1Bethel
1
(REU) ,
Melissa Hess
2
(RET) ,
Laura
3
Borecki ,
Dr. William
3
Eldridge
College, North Newton, KS 2Conestoga Valley Middle School 3Stroud Water Research Center, Avondale, PA
Abstract
Experimental Design
Results
Discussion
Environmental DNA (eDNA) surveillance is an increasingly
popular tool for detecting aquatic organisms that does not
require visual surveys. In many studies, this process has
shown to be successful at indicating the presence of
specific species (Muesnier et al. 2008). PCR primers were
previously developed to amplify small fragments of DNA
from several species of freshwater mussels including
Margaritifera margaritifera, Elliptio complanata, and
Pyganodon cataracta, and were shown to be effective in a
small stream setting (Eldridge and Borecki, pers. comm.).
However, there is still a limited understanding of how
eDNA moves through streams and water systems (Jane et
al. 2014). Understanding how the concentration of eDNA
changes over distance would provide some insight (Turner
et al. 2014). Real-time quantitative PCR (qPCR) is highly
sensitive (detection of 1 copy per uL is possible) and can
be used to quantify the amount of eDNA present (Wilcox
et al. 2013). We optimized qPCR conditions to ensure that
only the target sequence was amplified, a concern when
dealing with environmental samples. Optimal conditions
were: 1:10 dilution of template DNA, 65°C annealing
temperature, 1 mM MgCl2 and the addition of BSA. In an
outdoor flume, eDNA concentration increased over 25
meters downstream from four mussels, which may
indicate that mussels release DNA in pulses. In a natural
stream, eDNA was not detected 5m or more downstream
from the source which could be due to the presence of
natural PCR inhibitors. Applying the eDNA test to natural
setting, mussel DNA was detected in water samples from
one of three sites where they have not been observed by
visual surveys.
Four mussels were planted in a natural (small stream)
or artificial (outdoor flume) setting. In all experiments,
we allowed enough time for eDNA particles to
equilibrate downstream and then took 1L water samples
at various distances from the mussels.
Standard Curve for EcoCOI primer and Elliptio
complanata or Pyganodon cataracta
Based on validation tests for qPCR optimization (coefficient
of variation, R2, melt curve and DNA sequencing), qPCR
using the EcoCOI primers shows accurate and precise
quantification of mussel DNA. We expected that the
concentration of eDNA would decrease as it traveled further
away from the source because of settling (Newbold et
al.1982) but the flume experiment indicates that mussel
eDNA does not settle quickly. One possible explanation for
the concentration increase is that DNA is not released from
mussels at a constant rate. Instead, DNA could be released
in pulses (lots of DNA at one time) and we sampled different
parts of the DNA plume downstream. Future studies should
control for possible pulses of DNA release. One possible
way to determine eDNA transport while eliminating the
possibility of pulsed DNA is to conduct a DNA injection along
with rhodamine dye and to measure DNA in the water as the
dye travels downstream. The failure to detect DNA in the
small stream suggests that either DNA did not travel 5m or
that natural inhibitors prevented DNA amplification by PCR.
Natural inhibitors include humic acids, fulvic acids, melanin,
and polysaccharides which could all easily be present in the
small stream where we tested. qPCR also revealed that
mussel DNA was present in one site were they have not
been observed by visual surveys.
In this study, we optimized and used SYBR Green realtime qPCR. SYBR Green is a dye which fluoresces when
bound to double-stranded DNA. As more of the target
DNA is produced through PCR, more binding occurs and
thus there is an increase in fluorescence resulting in a
direct relationship between the change in fluorescence
and the initial quantity of the DNA template.
40
y = -2.2477x + 51.059
R² = 0.9509
35
Cт
30
25
20
y = -4.0429x + 46.011
R² = 0.9967
15
10
5
0
4
5
6
7
8
9
log10 (copy #)
Validation test for optimization
Outdoor flume
Water samples were also taken at sites where mussels
had not been previously detected by visual surveys. All
water samples were filtered using 47mm 1.5-micron
glass-fiber filters. The DNA was then extracted using
the DNEasy Blood & Tissue Kit (Qiagen, Valencia, CA
91355). qPCR was conducted on a ABI 7500 Fast RealTime PCR System using SYBR Green I and a melt
curve was generated to confirm that only one fragment
was amplified.
Margaritifera margaritifera,
the mussel species planted
for these experiments
Elliptio
Pyganodon
1.89%
Melt curve confirmed the presence of only
one PCR product
Melt curve
DNA sequencing confirmed that the intended
fragment was amplified
Sequencing
Using the formulas derived from the standard curves for each
primer, we were able to determine the number of DNA copies for
each of our unknown samples.
Change in Concentration over Distance (Flumes)
7.E+07
R² = 0.86028
6.E+07
5.E+07
4.E+07
3.E+07
2.E+07
qPCR Optimization
0.E+00
0
5
10
15
20
25
Distance from Source (m)
No eDNA was detected in Archie’s Branch (natural setting)
samples.
0 copies found
TEMPLATE DESIGN © 2008
www.PosterPresentations.com
qPCR using SYBR Green I (Applied Biosystems,
Carlsbad, CA 92008) was further optimized by testing the
intra-assay coefficient of variation (3 replicates per
sample), linearity of dilution (100 to 10-6) a melt curve and
sequencing.
30
Jane, S. F., Wilcox, T. M., McKelvey, K. S., Young, M. K., Schwartz,
M. K., Lowe, W. H., ... & Whiteley, A. R. (2014). Distance, flow,
and PCR inhibition: eDNA dynamics in two headwater streams.
Molecular Ecology Resources.
Meusnier, I., Singer, G. A., Landry, J.-F., Hickey, D. A., Hebert, P. D.,
& Hajibabaei, M. (2008). A universal DNA mini-barcode for
biodiversity analysis. BMC Genomics, 9(1), 214.
doi:10.1186/1471-2164-9-21
Newbold, J. D., O' Neill, R. V., Elwood, J. W., & Winkle, W. V. (1982).
Nutrient Spiralling in Streams: Implications for Nutrient Limitation
and Invertebrate Activity. The American Naturalist, 120, 628-652.
Turner, C. R., Barnes, M. A., Xu, C. C. Y., Jones, S. E., Jerde, C. L.,
& Lodge, D. M. (2014). Particle size distribution and optimal
capture of aqueous macrobial eDNA. bioRxiv. doi:10.1101/001941
Wilcox, T. M., McKelvey, K. S., Young, M. K., Jane, S. F., Lowe, W.
H., Whiteley, A. R., & Schwartz, M. K. (2013). Robust Detection of
Rare Species Using Environmental DNA: The Importance of
Primer Specificity. PLoS ONE, 8(3), e59520.
doi:10.1371/journal.pone.0059520
Acknowledgements
The authors acknowledge financial support from NSF EAR
1263212, a project entitled “Collaborate Research: REU/RET site
– Introducing Critical Zone Observatory science to students and
teachers”. BH was an REU intern at the SWRC during the
summer of 2014. Delaware Biotechnology Institute allowed the
use of their ABI 7500 Fast Real-time PCR System for this project.
The optimal PCR conditions were determined to be: 1:10
dilution of template DNA, 65° C annealing temperature,
1 mM MgCl2 and the presence of BSA.
Comparison with a known standard can be used to
determine the initial eDNA concentration in a sample.
Literature Cited
1.E+07
Prior to running the qPCR we optimized the PCR process
to make sure only the mussel target sequence was
amplified using the following conditions:
• Template DNA concentration: 1 uL template without
dilution,1:10 and 1:100 dilutions
• Annealing temperature: 60-70° C
• MgCl2 concentration: 1 mM and 1.5 mM MgCl2
• BSA: present or absent
http://www.bio-rad.com/en-us/applications-technologies/pcr-primer-probe-chemistries
11
R2 = 0.99672 (Elliptio), 0.95088 (Pyganodon)
Linearity of dilution
Stream: Archie’s Branch
10
Test Results
Coefficient of variation
# DNA Copies
How qPCR Works
45
0 copies found
Contact Information
190 copies found
Mussel DNA was detected in water samples from one of three
sites where they have not been observed by visual surveys
Bryce Hostetler
brycelhostetler[email protected]
Bethel College, North Newton, KS