Prepared in collaboration with the University of Minnesota

Detection of Environmental DNA of 

Bigheaded Carps in Samples Collected 

from Selected Locations in the St. Croix River 

and in the Mississippi River

Open-File Report 2013–1080

U.S. Department of the Interior

U.S. Geological Survey

i

Detection of Environmental DNA 

of Bigheaded Carps in Samples Collected 

from Selected Locations in the 

St. Croix River and in the Mississippi River

By Jon J. Amberg, Sunnie G. McCalla, Loren Miller, Peter Sorensen 

and Mark P. Gaikowski

Prepared in collaboration with the University of Minnesota

Open-File Report 2013–1080

U.S. Department of the Interior

U.S. Geological Survey
ii

U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2013

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Financial Disclosure

The analyses described in this interim report were fully funded by the Environment and Natural Resources Trust Fund 

through a grant to the University of Minnesota and through a Collaborative Agreement between the University of 

Minnesota and the U.S. Geological Survey. 

Suggested citation:

Amberg, J.J., McCalla, S.G., Miller, L., Sorensen, P., and Gaikowski, M.P., 2013, Detection of environmental DNA of 

Bigheaded Carps in samples collected from selected locations in the St. Croix River and in the Mississippi River: U.S. 

Geological Survey Open-File Report 2013–1080, 44 p.
iii

Contents

Background.....................................................................................................................................................1

Methods...........................................................................................................................................................2

Definition of Terms Used to Report the Detection of DNA.............................................................2

Sample Collection.................................................................................................................................3

DNA Extraction......................................................................................................................................3

PCR Analysis..........................................................................................................................................3

DNA Verification ...................................................................................................................................4

Results .............................................................................................................................................................4

Summary........................................................................................................................................................15

Acknowledgements.....................................................................................................................................15

References....................................................................................................................................................15

Appendixes 1–3............................................................................................................................................17

Appendix 1. Sample number, location of environmental sample collection, whether 

the sample was a cooler blank or filter positive control and notes on the 

presence of avian predators for each site. Additionally, the number of 

presumptive PCR-positives of the 8 PCR per sample, number of determinative 

PCR-positives of the 8 PCR per presumptive-positive sample and if the sample 

was confirmed positive for silver carp and/or bighead carp DNA. ..............................18

Appendix 2. Representative electrophoresis gels for the detection of the DNA 

of silver carp in samples taken from each site. Gels used for the detection of 

bighead carp were not included because bighead carp DNA was not 

detected. Representative gels for presumptive and determinative PCRpositives are included. .........................................................................................................27

Appendix 3. Sequences of determinative PCR-positive environmental samples amplified 

with the silver carp marker. The percent match of each sequence to published 

silver carp DNA sequences in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) is 

included following the sequence. A sample was determined “confirmed positive” 

only when both the forward and reverse sequences of that sample were greater 

than 95% similar to published silver carp sequences. ...................................................38

Figures

1. Detection of silver and bighead carp eDNA above the Coon Rapids Dam

 (Mississippi River) on September 18, 2012. Sample location of each 2-L water 

sample; circles indicate that the DNA of silver carp or bighead carp DNA 

was not detected ..........................................................................................................................7

2. Detection of silver and bighead carp eDNA below the Coon Rapids Dam 

(Mississippi River) on September 24, 2012. Sample location of each 2-L water 

sample; circles indicate that the DNA of silver carp or bighead carp DNA 

was not detected ..........................................................................................................................8

3. Detection of silver and bighead carp eDNA below Lock and Dam #1 

(Mississippi River) on September 25, 2012. Sample location of each 2-L water 

sample; circles indicate that the DNA of silver carp or bighead carp DNA 

was not detected ..........................................................................................................................9
iv

 4. Detection of silver and bighead carp eDNA above St. Croix Falls 
Detection of Environmental DNA of Bigheaded Carps 

in Samples Collected from Selected Locations in the 

St. Croix River and in the Mississippi River

By Jon J. Amberg1, Sunnie G. McCalla1, Loren Miller2,3, Peter Sorensen3 and Mark P. Gaikowski1

1

United States Geological Survey, Upper Midwest Environmental Sciences 

Center (UMESC), 2630 Fanta Reed Road, La Crosse, WI 54603 

2

Minnesota Department of Natural Resources, Department of Fisheries, 

Wildlife and Conservation Biology, University of Minnesota, 1980 Folwell 

Avenue, St. Paul, MN 55018

3

University of Minnesota (UMN), 

Background

The use of molecular methods, such as the detection of 

environmental deoxyribonucleic acid (eDNA), have become 

an increasingly popular tool in surveillance programs that 

monitor for the presence of invasive species in aquatic 

systems. One early application of these methods in aquatic 

systems was surveillance for DNA of Asian carps (specifically bighead carp Hypophthalmichthys nobilis and silver carp 

H. molitrix) in water samples taken from the Chicago Area 

Waterway System (CAWS; Jerde et al. 2011). Analysis of 

water samples from the CAWS to detect DNA of Asian carps 

has since been integrated into the Asian carp monitoring program managed by the Monitoring and Response Work Group, 

an interagency group established by the Asian Carp Regional 

Coordinating Committee. The U.S. Army Corps of Engineers 

(USACE) is presently responsible for the analysis of DNA in 

samples taken from the CAWS and has led the development 

and refinement of the methods and procedures used to process 

environmental samples to detect Asian carp DNA. Those 

methods are specified within the USACE “Quality Assurance 

Project Plan” or QAPP (USACE 2012).

The ability to identify DNA of a species in an environmental sample presents a potentially powerful tool to detect 

the leading edges of the invasion front of invasive animals 

such as Asian carp. Surveillance programs that incorporate 

eDNA monitoring have the potential for greater detection 

power because these sensitive analyses can presumably detect 

the presence of DNA in water even when the species is not 

abundant (Jerde et al. 2011). An initial eDNA surveillance 

effort was completed in Minnesota waters in 2011 after the 

capture of a bighead carp in the St. Croix River near Prescott, 

WI. Samples collected and analyzed for the presence of Asian 

carp DNA were expected to delineate the invasion front of 

bighead and silver carp in the Mississippi River and St. Croix 

River (Hickox et al. 2011; Hsu et al. 2011). The presence 

of silver carp DNA in samples collected in 2011 triggered a 

response by the Minnesota Department of Natural Resources 

(MN DNR) to intensively sample with conventional fishing 

gear areas where silver carp DNA was detected and to complete additional eDNA sampling and analysis. Although presumptive silver carp DNA was measured in several samples in 

the St. Croix and Mississippi Rivers, only two silver carp have 

since been captured in the Mississippi, and then downstream 

of the sampling locations, in the past two years (USGS 2013). 

Several bighead carp have also been captured by fishermen.

The environmental samples taken in 2011 for DNA 

analysis were processed by a private contractor using the 

markers identified in Jerde et al. (2011); PCR-positive samples 

were confirmed to contain the DNA of bighead carp or silver 

carp using a hybridization technique rather than sequencing 

as prescribed by the USACE QAPP. Subsequent to the 2011 

eDNA sampling and analysis efforts, multiple pathways (e.g. 

fish-eating birds, commercial fishing gear) have been identified that could transfer DNA within aquatic systems (USACE 

2013). The potential transfer of Asian carp DNA through vectors or fomites from locations where Asian carp are abundant 

to locations where they are absent or at low density might 

produce variable levels of basal DNA that would complicate 

interpretation of any silver carp or bighead carp DNA from a 

live fish. This study had several goals. The University, per its 

contract with the Environmental and Natural Resources Trust 

Fund (ENRTF), was primarily interested in determining if previous results from the Mississippi and St. Croix Rivers could 

be duplicated and were thus credible. It was secondarily interested in gaining critical insight into the nature of the QAPP 

technique so that recommendations for future work could be 

made to the MN DNR. We specifically sought to understand 

the potential confounding effects of these other pathways on 

this variation in background DNA by collecting water samples 

from (1) sites within the St. Croix River and the Mississippi 

River where the DNA of silver carp was previously detected, 
2 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

(2) sites considered to be free of Asian carp, and (3) a site 

known to have a large population of Asian carp. We also 

sought to establish a baseline Asian carp eDNA signature to 

which future eDNA sampling efforts could be compared. All 

samples described in this report were processed according 

to procedures of the USACE QAPP with seven exceptions 

designed to render the final analysis more definitive.

Water samples (Table 1) were collected from (1) sites 

similar to those sampled during the eDNA survey completed 

in 2011 (MN DNR 2011), (2) two Asian carp-free lakes that 

were expected to serve as negative controls, and (3) from the 

Mississippi River below Lock and Dam 19. Samples were 

collected below Lock and Dam 19 to serve as a positive 

control and to determine the DNA detection frequency, i.e., 

the frequency of detecting DNA in locations with high carp 

abundance. The negative control sites were expected to either 

not contain Asian carp DNA or to provide insight on potential 

background noise associated with the transfer of DNA from 

alternate pathways. 

Methods

Definition of Terms Used to Report the Detection 

of DNA

The following terms were used to describe the detection 

of Asian carp DNA in environmental samples through the use 

of conventional polymerase chain reaction (PCR) or Sanger 

sequencing.

PCR – Polymerase chain reaction

Negative control samples – samples that did not contain silver 

carp or bighead carp DNA (i.e. cooler blanks, well water 

laboratory controls and DNA-free PCR controls).

Positive control samples – samples that intentionally contained 

silver carp or bighead carp DNA.

Presumptive PCR-positive – bands between 150 to 300 bp for 

silver carp or 250 to 400 bp for bighead carp observed 

in any of the initial 8 PCR replicates processed from an 

environmental sample.

Determinative PCR-positive – bands between 150 to 300 bp 

for silver carp or 250 to 400 bp for bighead carp observed 

in any of the subsequent 8 PCR replicates prepared from 

an environmental sample determined to be presumptive 

PCR-positive.

Confirmed positive – appropriately sized bands from determinative PCR-positive environmental samples were 

submitted for Sanger sequencing. Resulting sequences 

were compared with published silver carp or bighead 

carp sequences by Basic Local Alignment Search Tool 

analysis (BLAST; Altschul et al. 1997); the sample was 

identified as a confirmed positive only if both the forward 

and reverse sequences (>150 bp in length) of the amplicon were ≥95% identical to published sequences of silver 

carp or bighead carp.

Table 1. Sites sampled and processed as part of the collaborative agreement between USGS-UMESC and the University of Minnesota. 

The coordinates given are the approximate centroid of the sampling location. Fifty water samples were taken and 3 cooler blanks were 

prepared at each sample location. Fifty samples of well water were taken and 25 samples of a slurry of silver carp and bighead carp 

mucus and feces were prepared to provide negative and positive control samples during DNA extraction and PCR analysis.

Water body Site description Reason for sampling

Reported in Hsu et 

al. (2012)

Date of sample 

collection

Location

(UTM Easting

/Northing)

Square Lake Washington County, MN Negative control N 10/01/2012 515976/4999913

Lake Riley Carver County, MN Negative control N 10/02/2012 459498/4964912

Mississippi River Above Coon Rapids Dam Surveillance Y 09/18/2012 474161/4999883

Below Coon Rapids Dam Surveillance Y 09/24/2012 475840/4998776

Below Lock and Dam 1 (Ford Dam) Surveillance Y 09/25/2012 485496/4971803

Below Lock and Dam 19 (Keokuk, IA) Positive control N 10/16/2012 637179/4471775

St. Croix River Above St. Croix Falls Dam Surveillance Y 09/17/2012 526806/5027129

Below St. Croix Falls Dam Surveillance Y 09/19/2012 527077/5031622

UMESC Well water (N=50) Negative control N 12/17/2012 NA

UMESC Silver carp slurry (N=25) Positive control N 12/17/2012 NA

UMESC Bighead carp slurry (N=25) Positive control N 3/1/2013 NA
Methods 3

Sample Collection

Fifty, 2-L surface water samples (surface to approximately 5 cm below surface) were collected from each of eight 

sites (Table 1) by targeting specific areas (eddies, below and 

around structures, confluence of tributaries, etc.) as well as 

shore samples and transects attempting to get a dispersed 

coverage of the sampling sites (Figures 1-8). Samples were 

processed according to procedures specified in the USACE 

QAPP by personnel from MN DNR and UMN, except that 

sterile, disposable funnels (Pall MicroFunnel 300) were used 

instead of a reusable magnetic filter funnel that must be sterilized between samples. These funnels were identical to those 

used in 2011 (Hickox et al. 2011; Hsu et al 2012). Cooler 

blanks (n=3) were prepared at each site concurrent with the 

collection of the 50 environmental samples. The presence of 

avian predators (e.g. fish-eating birds) was noted during collection of environmental samples (Appendix 1). No attempt is 

made by this report to link the presence of Asian carp DNA to 

potential avian transport of DNA from one location to another. 

All other sampling procedures, including the collection and 

handling of appropriate controls (i.e. cooler blanks), followed 

the procedures specified in the USACE QAPP. Each sample 

was filtered through a 1.5 μm glass-fiber filter; filters were 

placed in labeled, 50-mL conical centrifuge tubes and immediately stored at 20°C. Filters were transported on dry ice to 

UMESC where they were then stored at -80°C until processed 

to extract DNA and complete PCR analysis.

Laboratory positive control samples consisted of 25 samples of silver carp slurry prepared from silver carp mucus and 

fecal material and 25 samples of bighead carp slurry prepared 

from bighead carp mucus and fecal material. Laboratory negative control samples consisted of 50 samples of UMESC well 

water. All laboratory positive and negative control samples 

were prepared at UMESC by UMESC personnel. 

DNA Extraction

The DNA adhering to filters was extracted using the MO 

BIO PowerWater DNA Isolation Kit (MO BIO Laboratories, 

Inc., Carlsbad, CA) according to the procedures specified 

in the USACE QAPP with certain modifications. The DNA 

extracted from sample filters was stored at -20°C until PCR 

analysis. The modifications of the USACE QAPP procedures 

were made to improve rigor in our sample analyses and 

decrease the potential for contamination and were as follows:

•	 Each extraction kit (one kit contained the requisite consumables to complete 100 extractions) was 

subdivided on receipt at UMESC into separate sets 

sufficient to complete 25 extractions. This reduced 

the risk of contamination of the consumables and 

reduced the risk that contamination of consumables 

might impact multiple sample sets. 

•	 Extractions were completed on sets of 20 unknown 

samples. All preparatory surfaces were sterilized with 

10% hypochlorous acid solution (bleach) or exposure 

to UV light (>15 minutes) between each set of 20 

unknown sample extractions. 

•	 Extracting DNA from 3 or more filters in a single 

PowerWater extraction increased the rate of failure of 

the extraction process (USACE 2013). For samples 

with 2 or more filters, DNA was extracted from no 

more than 2 filters within an extraction vessel. This 

modification was recently incorporated into the procedures specified in the USACE QAPP.

•	 A positive silver carp control sample and a negative 

control sample were processed with each batch of 20 

extractions. This deviated from the USACE QAPP, 

which requires one positive and one negative control 

sample for every 25 extractions.

•	 Samples that contained Asian carp DNA following initial PCR (presumptive PCR-positive) were 

subjected to a second PCR analysis (in octuplet) to 

confirm the presence of Asian carp DNA. Samples 

were only considered determinative PCR-positive if 

Asian carp DNA was amplified in at least one of the 

octuplet replicates in the second PCR analysis. This 

deviated from the USACE QAPP that dropped the 

processing of the second PCR analysis. Completing 

the second PCR analysis provided a more rigorous 

approach to eliminate non-specific amplification 

of DNA sequences of lengths similar to the target 

amplicon.

•	 The DNA of 50% of the presumptive PCR-positives 

(>1 positive replicate of the 8 PCR replicates) from 

below Lock and Dam 19 were re-amplified. All of the 

determinative PCR-positives were sequenced. This 

deviated from the USACE QAPP which specifies 

that 5% of positive samples from known Asian carp 

waters be sequenced.

PCR Analysis

The presence of silver carp DNA or bighead carp 

DNA was determined by PCR using the following species specific markers: silver carp, forward CCTGARAAAAGARKTRTTCCACTATAA, reverse GCCAAATGCAAGTAATAGTTCATTC; bighead carp, forward 

TAACTTAAATAAACAGATTA, reverse TAAAAGAATGCTCGGCATGT. These markers (originally reported by 

Jerde et al. 2011) are presently used in the Asian carp eDNA 

surveillance program applied in the CAWS. Each sample 

was processed in octuplet by PCR for each species. Each run 

contained a positive and a negative PCR control, as well as 

a laboratory positive and a negative control sample. All PCR 
4 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

reactions (25 μL) were prepared using the reagents specified 

in the USACE QAPP. The PCR conditions used for the silver 

carp marker was initial denaturation at 94°C for 10 minutes 

followed by 45 cycles of 94°C for 1 minute then 50°C for 1 

minute then 72°C for 1.5 minutes; there was a final extension 

at 72°C for 7 minutes after the 45 cycles. The PCR conditions 

used for the bighead carp marker were the same except that a 

temperature of 52°C was used instead of 50°C. The PCR products were held at 4°C until the plate was removed from the 

thermocycler. All original DNA extracts were stored at -20°C. 

The PCR products were visualized by electrophoresis on a 2% 

agarose gel, stained with GelRedTM (Biotium Inc., Hayward, 

CA, USA). The presence of the appropriate band was visualized under ultraviolet light. Any sample in which DNA of the 

appropriate amplicon length (i.e. ~190 bp for silver carp and 

~310 bp for bighead carp) was present in one or more of the 

8 PCR replicates in the initial PCR analysis was considered 

presumptive PCR-positive. Occasionally, bands of a similar 

length to that of the targeted sequences can be detected, likely 

due to non-specific amplification. If a band of the appropriate 

length was observed in any of the 8 PCR replicates of a DNA 

extract, the sample was re-analyzed by PCR using the process 

described above except that only a positive and a negative 

PCR control were used. Any sample in which DNA of the 

appropriate amplicon length was present in one or more of the 

8 PCR replicates in the second PCR analysis was considered 

determinative PCR-positive. All samples considered presumptive PCR-positive were re-analyzed within 6 weeks of the 

original PCR analysis.

DNA Verification

The DNA of all determinative PCR-positive samples 

from any location other than below Lock and Dam 19 was 

eluted from the agarose gel using a QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) and cleaned using a 

QIAquick PCR Purification Kit (QIAGEN Inc., Valencia, CA). 

The DNA from 50% of the determinative PCR-positives (17 

of 34) from below Lock and Dam 19 was re-analyzed and 

any subsequent determinative PCR-positives (16 of 17) were 

eluted as described above. The eluted DNA was transported 

to the University of Minnesota Biomedical Genomics Center 

(St. Paul, MN) for Sanger sequencing (forward and reverse 

sequencing). The resulting sequences were compared using 

BLAST analysis to published sequences in GenBank (http://

www.ncbi.nlm.nih.gov/genbank/). A sample was identified 

as a confirmed positive only if both the forward and reverse 

sequences (>150 bp in length) of the amplicon were ≥95% 

identical to published sequences of silver carp or bighead carp.

Results

A total of 596 DNA extractions were performed from 

400 environmental samples, 25 silver carp positive control 

samples, 25 bighead carp positive control samples and 50 

negative control samples. Contamination was not observed 

in any negative control sample (i.e., DNA was not detected 

in any laboratory negative control sample or negative PCR 

control) or in any cooler blank. The DNA of Asian carp was 

detected in all laboratory positive control samples and positive 

PCR controls. 

The location and the detection of silver carp DNA is 

reported in Table 2, Figures 1-8 and Appendix 1. Thirty four of 

the 50 environmental samples below Lock and Dam 19 were 

considered to be presumptive PCR-positive for the presence of 

silver carp DNA. Sixteen of 17 of these environmental samples were found to be determinative PCR-positive; all determinative PCR-positive samples were confirmed positive for silver carp DNA by Sanger sequencing (Appendix 3). The DNA 

extracted from the positive control site (below Lock and Dam 

19) appeared to be of a higher quality (cleaner bands) and was 

visually more similar to the bands observed in extracts from 

the positive extraction and PCR controls than those observed 

from the negative control sites or surveillance sites (Appendix 

2). Only three environmental samples, two from above the St. 

Croix Falls Dam and one from Square Lake, were found to be 

determinative PCR-positive for silver carp DNA. The DNA 

sequences of the determinative PCR products of these three 

environmental samples were identified as not being silver carp 

DNA (Appendix 3). The number of samples processed and the 

portion of presumptive PCR-positive, determinative PCRpositive and confirmed positive samples in which silver carp 

DNA was detected are reported in Table 2; results of samples 

processed to detect bighead carp DNA are reported in Table 3. 

Results of Sanger sequencing of determinative PCR-positive 

environmental samples are summarized in Appendix 3. 

The location and the detection of bighead carp DNA is 

reported in Table 3, Figures 1-8 and Appendix 1. Bighead carp 

DNA was not detected in any of the environmental samples, 

including those collected below Lock and Dam 19 where 

bighead carp are often captured by commercial fishers (Iowa 

Department of Natural Resources, 2012).
Results 5

Table 2. Number of presumptive PCR-positive, determinative PCR-positive and confirmed positive (by PCR product sequencing) 

samples in which the DNA of silver carp was detected. 

Site Sample type Total # samples Presumptive 

PCR-positive

Determinative 

PCR-positive

Confirmed 

positives

Positive Control Site

Below Lock and Dam 19 River water 50 34 16 of 17* 16 of 16*

Cooler blank 3 0 -- --

Negative Control Sites

Square Lake Lake water 50 11 1 0

Cooler blank 3 0 -- --

Lake Riley Lake water 50 6 0 --

Cooler blank 3 0 -- --

Mississippi River Test Sites

Above Dam at Coon Rapids River water 50 2 0 --

Cooler blank 3 0 -- --

Below Dam at Coon Rapids River water 50 2 0 --

Cooler blank 3 0 -- --

Below Lock and Dam 1 River water 50 12 0 --

Cooler blank 3 0 -- --

St. Croix River Test Sites

Above St. Croix Falls Dam River water 50 6 2 0

Cooler blank 3 0 -- --

Below St. Croix Falls Dam River water 50 10 0 --

Cooler blank 3 0 -- --

Laboratory Controls

Negative control Water 50 0 -- --

Silver carp slurry (positive control) Slurry 25 25 NA NA

*

The QAPP recommends that 5% of the positives be sampled unless ecologically important. Since the site below Lock and Dam 19 has a population of 

bighead carp and silver carp that represent a significant portion of the commercial harvest of that water body, it was classified as a positive control. Nonetheless, 

50% of the presumptive PCR-positives were re-analyzed and 100% of the amplicons of the determinative PCR-positive samples sequenced.
6 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Table 3. Number of presumptive PCR-positive, determinative PCR-positive and confirmed positive (by PCR product sequencing) 

samples in which the DNA of bighead carp was detected. 

Site Sample type Total # samples Presumptive 

PCR-positive

Determinative 

PCR-positive

Confirmed 

positives

Positive Control Site

Below Lock and Dam 19 River water 50 0 -- --

Cooler blank 3 0 -- --

Negative Control Sites

Square Lake Lake water 50 0 -- --

Cooler blank 3 0 -- --

Lake Riley Lake water 50 0 -- --

Cooler blank 3 0 -- --

Mississippi River Test Sites

Above Dam at Coon Rapids River water 50 0 -- --

Cooler blank 3 0 -- --

Below Dam at Coon Rapids River water 50 0 -- --

Cooler blank 3 0 -- --

Below Lock and Dam 1 River water 50 1 0 --

Cooler blank 3 0 -- --

St. Croix River Test Sites

Above St. Croix Falls Dam River water 50 0 -- --

Cooler blank 3 0 -- --

Below St. Croix Falls Dam River water 50 0 -- --

Cooler blank 3 0 -- --

Laboratory Controls

Negative control Well water 50 0 -- --

Bighead carp slurry (positive 

control)

Slurry 25 25 NA NA
Results 7

Figure 1. Detection of silver and bighead carp eDNA above the Coon Rapids Dam (Mississippi River) on September 18, 2012. 

Sample location of each 2-L water sample; circles indicate that the DNA of silver carp or bighead carp DNA was not detected.
8 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Figure 2. Detection of silver and bighead carp eDNA below the Coon Rapids Dam (Mississippi River) on September 24, 2012. 

Sample location of each 2-L water sample; circles indicate that the DNA of silver carp or bighead carp DNA was not detected.
Results 9

Figure 3. Detection of silver and bighead carp eDNA below Lock and Dam #1 (Mississippi River) on September 25, 2012. 

Sample location of each 2-L water sample; circles indicate that the DNA of silver carp or bighead carp DNA was not detected.
10 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Figure 4. Detection of silver and bighead carp eDNA above St. Croix Falls (St. Croix River) on September 17, 2012. Sample location of 

each 2-L water sample; circles indicate that the DNA of silver carp or bighead carp DNA was not detected.
Results 11

Figure 5. Detection of silver and bighead carp eDNA below St. Croix Falls (St. Croix River) on September 19, 2012. Sample location of 

each 2-L water sample; circles indicate that the DNA of silver carp or bighead carp DNA was not detected.
12 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Figure 6. Detection of silver and bighead carp eDNA below Lock and Dam 19 (Mississippi River) on October 16, 2012. Sample location 

of each 2-L water sample; circles indicate that silver carp DNA was not detected, “X” indicate presumptive PCR-positive samples that 

were not processed for determinative PCR or sequencing, triangles indicate samples confirmed positive by sequencing. The DNA of

bighead carp was not detected in any of the samples.
Results 13

Figure 7. Detection of silver and bighead carp eDNA in Square Lake on October 1, 2012. Sample location of each 2-L water sample; 

circles indicate that the DNA of silver carp or bighead carp DNA was not detected.
14 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Figure 8. Detection of silver and bighead carp eDNA in Lake Riley on October 2, 2012. Sample location of each 2-L water sample; 

circles indicate that the DNA of silver carp or bighead carp DNA was not detected.
References 15

Summary

1. The DNA of bighead carp and silver carp was not detected 

in environmental samples (n=50 per site) collected above 

and below St. Croix Falls Dam on the St. Croix River, 

above and below the Coon Rapids Dam and below Lock 

and Dam 1 on the Mississippi River, and from two negative control lakes, Square Lake and Lake Riley. 

2. The DNA of silver carp was detected in environmental 

samples (n=50) collected below Lock and Dam 19 at 

Keokuk, Iowa, a reach of the river with high silver carp 

abundance. Not all samples were presumptive PCR-positive, but the portion (68%) was similar to that reported 

in the scientific literature for an abundant species (e.g., 

bighead carp and silver carp, Jerde et al. 2011; common 

carp Cyprinus carpio; Mahon et al. 2013). All determinative PCR-positive samples were confirmed to be silver 

carp DNA.

3. The DNA of bighead carp was not detected in environmental samples (n=50) collected below Lock and Dam 

19, a reach of the river known to have bighead carp.

4. The previous reported detections of the DNA of silver 

carp in samples collected in 2011 (Hickox et al. 2011; Hsu 

et al. 2012) were not replicated in this study. Those previous studies used a hybridization technique as a final confirmation that a PCR-positive sample contained silver carp 

DNA. The USACE QAPP followed here uses sequencing 

(forward and reverse) to confirm whether the DNA in a 

PCR-positive sample is/is not the DNA of bighead carp or 

silver carp. 

5. Sequencing (forward and reverse) PCR-positive samples 

provides the greatest certainty that the DNA of the target 

species is present and should be used in any location 

where the target species is thought to be rare or absent 

(i.e., all locations other than positive control sites). Any 

monitoring program using current markers for Asian carp 

(e.g. Jerde et al. 2011) should use sequencing as the final 

confirmation step to assure comparability of results across 

years and locations.

Acknowledgements

This project could not have been completed without 

Byron Karns of the National Park Service, who provided 

resources, equipment and time for the collection and processing of field water samples. James Lamer of Western Illinois 

University provided valuable insight, guidance and resources 

for samples collected below Lock and Dam 19. Finally, this 

project was funded through reimbursable agreement number 

13EMN00000002 with the University of Minnesota, which 

received financial support from the Environment and Natural 

Resources Trust Fund.

References

Altschul, SF, TL Madden, AA Schaffer, JH Zhang, Z Zhang, 

W Miller and DJ Lipman. 1997. Gapped BLAST and 

PSI-BLAST: a new generation of protein database search 

programs. Nucleic Acids Research. 25(17): 3389–3402. 

Hickox, T, F-C Hsu, R Wong, and T Brito-Robinson. 2011. 

eDNA Surveillance of Asian Carp on the St. Croix and Mississippi Rivers. Final Report 11 August 2011. Report to the 

Minnesota Department of Natural Resources, St. Paul.

Hsu, F-C., R Wong, T Brito-Robinson, and M Hayes. 2012. 

eDNA Surveillance of Asian Carp in Minnesota waters, 

Fall 2011. Report to the Minnesota Department of Natural 

Resources, St. Paul.

Iowa Department of Natural Resources. 2012. Commercial 

Fish Report for Iowa Boundary Waters of the Mississippi 

and Missouri Rivers. 

Jerde, CL, AR Mahon, WL Chadderton, and DM Lodge. 2011. 

‘Sight-Unseen’ Detection of Rare Aquatic Species Using 

Environmental DNA. Conservation Letters. 4: 150–157. 

DOI: 10.1111/j.1755-263X.2010.00158.x.

Mahon, AR, CL Jerde, M Galaska, JL Bergner, WL Chadderton, DM. Lodge, ME Hunter, LG Nico. 2013. Validation of 

eDNA Surveillance Sensitivity for Detection of Asian Carps 

in Controlled and Field Experiments. PLoS ONE 8(3): 

e58316. doi:10.1371/journal.pone.0058316

U.S. Army Corps of Engineers. 2012. Quality Assurance 

Project Plan: eDNA Monitoring of Invasive Asian Carp in 

the Chicago Area Waterway System. Retrieved from http://

www.asiancarp.us/documents/USACE-eDNA-QAPP.pdf

U.S. Army Corps of Engineers. 2013. Environmental DNA 

Calibration Study: Interim Technical Review Report. 

Retrieved from http://www.asiancarp.us/documents/

ECALS_INTERIM.pdf

U.S. Geological Survey. 2013. Nonindigenous Aquatic Species 

Database. Available from: http://nas.er.usgs.gov. Accessed 

05 March 2013.
16 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River
Appendixes 1–3 17

Appendixes 1–3

Appendix 1. Sample number, location of environmental sample collection, whether the sample 

was a cooler blank or filter positive control and notes on the presence of avian predators for 

each site. Additionally, the number of presumptive PCR-positives of the 8 PCR per sample, 

number of determinative PCR-positives of the 8 PCR per presumptive-positive sample and if the 

sample was confirmed positive for silver carp and/or bighead carp DNA.

Appendix 2. Representative electrophoresis gels for the detection of the DNA of silver carp 

in samples taken from each site. Gels used for the detection of bighead carp were not included 

because bighead carp DNA was not detected. Representative gels for presumptive and determinative PCR-positives are included.

Appendix 3. Sequences of determinative PCR-positive environmental samples amplified 

with the silver carp marker. The percent match of each sequence to published silver carp 

DNA sequences in GenBank (http://www.ncbi.nlm.nih.gov/genbank/ ) is included following 

the sequence. A sample was determined “confirmed positive” only when both the forward 

and reverse sequences of that sample were greater than 95% similar to published silver carp 

sequences. 
18 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Appendix 1. Sample number, location of environmental sample collection, whether the sample was 

a cooler blank or filter positive control and notes on the presence of avian predators for each site. 

Additionally, the number of presumptive PCR-positives of the 8 PCR per sample, number of determinative 

PCR-positives of the 8 PCR per presumptive-positive sample and if the sample was confirmed positive for 

silver carp and/or bighead carp DNA.
Appendixes 1–3 19

Date collected: September 17, 2012

Location: St. Croix River above St. Croix Falls Dam

Silver Carp Bighead Carp

NOTES

Presumptive 

PCR-PositivesDeterminative PCR-Positives

Confirmed

Presumptive 

PCR-PositivesDeterminative PCR-Positives

Confirmed

ID Easting Northing Blank

No. of 

Filters

001 0525454 5034558 ----- 2 0 0 ----- 0 0 ----- eagle eating fish nearby

002 0525463 5034485 ----- 2 0 0 ----- 0 0 ----- eagle eating fish nearby

003 0525426 5034375 ----- 2 0 0 ----- 0 0 ----- osprey flew over

004 0525378 5034242 ----- 2 0 0 ----- 0 0 -----

005 0525584 5034178 ----- 2 1 0 ----- 0 0 -----

006 0525746 5034269 ----- 2 0 0 ----- 0 0 -----

007 0525905 5034279 BLANK 2 0 0 ----- 0 0 -----

008 0525905 5034279 ----- 2 0 0 ----- 0 0 -----

009 0525900 5034183 ----- 2 0 0 ----- 0 0 -----

010 0525874 5034037 ----- 2 2 0 ----- 0 0 ----- gull flew over

011 0526335 5033892 ----- 2 0 0 ----- 0 0 -----

012 0526499 5034027 ----- 2 0 0 ----- 0 0 -----

013 0526994 5033806 ----- 2 0 0 ----- 0 0 -----

014 0526974 5033688 ----- 2 0 0 ----- 0 0 -----

015 0526980 5033565 ----- 2 0 0 ----- 0 0 -----

016 0527222 5032983 ----- 2 1 1 NO 0 0 -----

017 0527495 5032869 ----- 2 0 0 ----- 0 0 -----

018 0527611 5032523 ----- 2 0 0 ----- 0 0 -----

019 0527533 5032439 ----- 2 0 0 ----- 0 0 -----

020 0527500 5032240 ----- 2 1 0 ----- 0 0 -----

021 0527551 5031947 ----- 2 0 0 ----- 0 0 ----- eagle flying nearby

022 0527736 5031932 ----- 2 0 0 ----- 0 0 -----

023 0527781 5031704 ----- 2 0 0 ----- 0 0 -----

024 0527680 5031676 ----- 2 0 0 ----- 0 0 -----

025 0527571 5031702 ----- 2 0 0 ----- 0 0 -----

026 0527537 5031489 ----- 2 0 0 ----- 0 0 -----

027 0527444 5031355 ----- 2 0 0 ----- 0 0 -----

028 0527638 5031285 ----- 2 0 0 ----- 0 0 -----

029 0527599 5030986 ----- 2 0 0 ----- 0 0 ----- kingfisher

030 0527503 5030989 ----- 2 0 0 ----- 0 0 -----

031 0527371 5030977 ----- 2 0 0 ----- 0 0 -----

032 0527345 5030823 ----- 2 0 0 ----- 0 0 -----

033 0527585 5030737 ----- 2 0 0 ----- 0 0 -----

034 0527603 5030613 ----- 2 0 0 ----- 0 0 -----

035 0527503 5030557 ----- 2 0 0 ----- 0 0 -----

036 0527377 5030423 ----- 2 0 0 ----- 0 0 -----

037 0527368 5030274 ----- 2 0 0 ----- 0 0 -----

038 0527489 5030104 ----- 2 0 0 ----- 0 0 -----

039 0527547 5029975 ----- 2 0 0 ----- 0 0 ----- cormorant 

040 0527398 5029906 BLANK 1 0 0 ----- 0 0 -----

041 0527398 5029906 ----- 2 0 0 ----- 0 0 -----

042 0527240 5029786 ----- 2 0 0 ----- 0 0 -----

043 0527235 5029717 ----- 2 1 1 NO 0 0 -----

044 0527483 5029660 ----- 2 0 0 ----- 0 0 -----

045 0527498 5029616 ----- 2 0 0 ----- 0 0 -----

046 0527498 5029450 ----- 2 0 0 ----- 0 0 -----

047 0527413 5029397 ----- 2 0 0 ----- 0 0 -----

048 0527272 5029356 BLANK 1 0 0 ----- 0 0 -----

049 0527272 5029356 ----- 2 0 0 ----- 0 0 -----

050 0527280 5029283 ----- 2 0 0 ----- 0 0 -----

051 0527256 5029134 ----- 2 0 0 ----- 0 0 -----

052 0527339 5028935 ----- 2 0 0 ----- 0 0 -----

053 0527475 5028017 ----- 2 3 0 ----- 0 0 -----

054 ----- ----- Filter + 2 3 ----- ----- ----- ----- -----
20 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Date collected: September 18, 2012

Location: Mississippi River Above Coon Rapids Dam

Silver Carp Bighead Carp

NOTES

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

ID Easting Northing Blank

No. of 

Filters

001 472878 5000841 ----- 2 1 0 ----- 0 0 -----

002 473012 5000730 ----- 2 0 0 ----- 0 0 -----

003 473146 5000639 ----- 2 0 0 ----- 0 0 -----

004 473368 5000482 ----- 3 0 0 ----- 0 0 -----

005 473479 5000400 ----- 2 0 0 ----- 0 0 -----

006 472818 5000802 ----- 2 0 0 ----- 0 0 -----

007 473106 5000555 ----- 2 0 0 ----- 0 0 -----

008 473379 5000364 ----- 2 0 0 ----- 0 0 -----

009 472695 5000771 ----- 2 0 0 ----- 0 0 -----

010 472864 5000631 ----- 2 0 0 ----- 0 0 -----

011 472970 5000540 ----- 2 0 0 ----- 0 0 -----

012 473181 5000390 BLANK 1 0 0 ----- 0 0 -----

013 473181 5000390 ----- 2 0 0 ----- 0 0 -----

014 473320 5000318 ----- 2 0 0 ----- 0 0 -----

015 473514 5000172 ----- 2 0 0 ----- 0 0 -----

016 473639 5000083 ----- 2 2 0 ----- 0 0 -----

017 473835 4999951 ----- 2 0 0 ----- 0 0 -----

018 473973 4999850 ----- 2 0 0 ----- 0 0 -----

019 474120 4999722 ----- 2 0 0 ----- 0 0 -----

020 473673 5000182 ----- 2 0 0 ----- 0 0 -----

021 474023 4999986 ----- 2 0 0 ----- 0 0 -----

022 473557 5000350 ----- 2 0 0 ----- 0 0 -----

023 473736 5000250 ----- 2 0 0 ----- 0 0 -----

024 473848 5000206 ----- 2 0 0 ----- 0 0 -----

025 474150 5000077 BLANK 1 0 0 ----- 0 0 -----

026 474150 5000077 ----- 2 0 0 ----- 0 0 -----

027 474334 4999911 ----- 2 0 0 ----- 0 0 ----- eagle flying nearby

028 474405 4999855 ----- 2 0 0 ----- 0 0 -----

029 474501 4999749 ----- 2 0 0 ----- 0 0 -----

030 474599 4999609 ----- 2 0 0 ----- 0 0 -----

031 474662 4999520 ----- 2 0 0 ----- 0 0 -----

032 474716 4999446 ----- 2 0 0 ----- 0 0 -----

033 474347 4999732 ----- 2 0 0 ----- 0 0 -----

034 474182 4999641 ----- 2 0 0 ----- 0 0 -----

035 474257 4999557 ----- 4 0 0 ----- 0 0 -----

036 474308 4999492 ----- 2 0 0 ----- 0 0 -----

037 474408 4999343 ----- 2 0 0 ----- 0 0 -----

038 474552 4999221 ----- 2 0 0 ----- 0 0 -----

039 474608 4999188 ----- 2 0 0 ----- 0 0 -----

040 474749 4999125 ----- 4 0 0 ----- 0 0 -----

041 474966 4999057 ----- 2 0 0 ----- 0 0 -----

042 474125 4999995 ----- 2 0 0 ----- 0 0 -----

043 474249 4999939 ----- 2 0 0 ----- 0 0 -----

044 474893 4999194 BLANK 1 0 0 ----- 0 0 -----

045 474893 4999194 ----- 2 0 0 ----- 0 0 -----

046 474812 4999369 ----- 2 0 0 ----- 0 0 -----

047 474892 4999325 ----- 2 0 0 ----- 0 0 -----

048 475044 4999279 ----- 2 0 0 ----- 0 0 -----

049 475209 4999280 ----- 2 0 0 ----- 0 0 -----

050 475415 4999239 ----- 2 0 0 ----- 0 0 -----

051 475535 4999194 ----- 2 0 0 ----- 0 0 ----- gull over dam

052 475619 4999319 ----- 2 0 0 ----- 0 0 -----

053 475622 4999243 ----- 2 0 0 ----- 0 0 -----

054 ----- ----- Filter + 2 5 ----- ----- ----- ----- -----
Appendixes 1–3 21

Date collected: September 19, 2012

Location: St. Croix River below St. Croix Falls Dam

Silver Carp Bighead Carp

NOTES

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

ID Easting Northing Blank

No. of 

Filters

001 0527473 5028738 ----- 2 1 0 ----- 0 0 -----

002 0527523 5028725 ----- 2 0 0 ----- 0 0 -----

003 0527512 5028722 ----- 2 2 0 ----- 0 0 -----

004 0527459 5028717 BLANK 2 0 0 ----- 0 0 ----- many crows above

005 0527459 5028717 ----- 2 1 0 ----- 0 0 -----

006 0527434 5028668 ----- 2 0 0 ----- 0 0 -----

007 0527432 5028613 ----- 2 1 0 ----- 0 0 -----

008 0527409 5028552 ----- 2 0 0 ----- 0 0 -----

009 0527373 5028074 ----- 2 0 0 ----- 0 0 ----- eagle

010 0527365 5028020 ----- 2 2 0 ----- 0 0 -----

011 0527320 5027686 ----- 1 0 0 ----- 0 0 -----

012 0527328 5027969 ----- 2 0 0 ----- 0 0 -----

013 0527311 5027945 ----- 2 2 0 ----- 0 0 -----

014 0527298 5027895 ----- 2 0 0 ----- 0 0 -----

015 0527271 5027822 ----- 2 0 0 ----- 0 0 -----

016 0527585 5028608 ----- 3 0 0 ----- 0 0 ----- crows above, feces 

017 0527562 5028535 ----- 2 1 0 ----- 0 0 -----

018 0527606 5028431 ----- 2 1 0 ----- 0 0 -----

019 0527614 5028370 ----- 2 0 0 ----- 0 0 -----

020 0527610 5028271 ----- 2 0 0 ----- 0 0 -----

021 0527599 5028037 ----- 2 0 0 ----- 0 0 -----

022 0527532 5027979 ----- 2 0 0 ----- 0 0 -----

023 0527608 5028184 ----- 2 0 0 ----- 0 0 -----

024 0527390 5027526 ----- 2 0 0 ----- 0 0 -----

025 0527342 5027481 ----- 2 0 0 ----- 0 0 -----

026 0527360 5027428 ----- 2 0 0 ----- 0 0 -----

027 0527352 5027362 ----- 2 0 0 ----- 0 0 -----

028 0527378 5027352 ----- 2 0 0 ----- 0 0 -----

029 0527427 5027348 ----- 2 0 0 ----- 0 0 -----

030 0527316 5027301 ----- 2 0 0 ----- 0 0 -----

031 0527174 5027206 ----- 2 0 0 ----- 0 0 -----

032 0527169 5027167 ----- 2 0 0 ----- 0 0 -----

033 0527000 5027114 ----- 2 0 0 ----- 0 0 -----

034 0526882 5027080 ----- 2 0 0 ----- 0 0 -----

035 0526619 5027030 BLANK 1 0 0 ----- 0 0 -----

036 0526619 5027030 ----- 2 0 0 ----- 0 0 -----

037 0526365 5026992 ----- 2 0 0 ----- 0 0 ----- turkey vulture

038 0526196 5026750 ----- 2 0 0 ----- 0 0 -----

039 0526103 5026332 ----- 2 0 0 ----- 0 0 -----

040 0525905 5026121 ----- 2 0 0 ----- 0 0 -----

041 0525798 5026117 ----- 2 1 0 ----- 0 0 -----

042 0525661 5025997 ----- 2 0 0 ----- 0 0 -----

043 0525526 5025755 ----- 2 0 0 ----- 0 0 -----

044 0525530 5025469 ----- 2 0 0 ----- 0 0 -----

045 0525420 5025428 ----- 2 0 0 ----- 0 0 -----

046 0525301 5025355 ----- 2 0 0 ----- 0 0 -----

047 0525196 5025069 ----- 2 0 0 ----- 0 0 -----

048 0525104 5024767 ----- 2 0 0 ----- 0 0 -----

049 0525151 5024513 ----- 2 0 0 ----- 0 0 -----

050 0525172 5024463 ----- 2 0 0 ----- 0 0 -----

051 0525223 5024321 BLANK 1 0 0 ----- 0 0 -----

052 0525223 5024321 ----- 2 0 0 ----- 0 0 -----

053 0525113 5024353 ----- 2 1 0 ----- 0 0 -----

054 ----- ----- Filter + 2 0 ----- ----- ----- ----- -----
22 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Date collected: September 24, 2012

Location: Mississippi River below Coon Rapids Dam

Silver Carp Bighead Carp

NOTES

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

ID Easting Northing Blank

No. of 

Filters

001 475672 4999208 ----- 2 0 0 ----- 0 0 -----

002 475683 4999174 ----- 2 0 0 ----- 0 0 -----

003 475691 4999129 ----- 2 0 0 ----- 0 0 -----

004 475730 4999160 ----- 2 0 0 ----- 0 0 -----

005 475761 4999165 ----- 2 0 0 ----- 0 0 -----

006 475811 4999188 ----- 2 0 0 ----- 0 0 -----

007 475703 4999109 ----- 2 0 0 ----- 0 0 -----

008 475651 4999042 ----- 4 0 0 ----- 0 0 -----

009 475669 4999035 ----- 4 0 0 ----- 0 0 -----

010 475694 4999027 ----- 6 0 0 ----- 0 0 -----

011 475720 4998986 ----- 2 0 0 ----- 0 0 -----

012 475748 4998959 ----- 2 0 0 ----- 0 0 -----

013 475757 4999025 ----- 4 1 0 ----- 0 0 -----

014 475802 4998977 ----- 2 1 0 ----- 0 0 -----

015 475829 4998941 ----- 2 0 0 ----- 0 0 ----- eagle flying

016 475810 4998894 ----- 2 0 0 ----- 0 0 -----

167 475780 4998854 ----- 2 0 0 ----- 0 0 -----

168 475914 4998916 ----- 2 0 0 ----- 0 0 -----

019 476035 4998838 ----- 2 0 0 ----- 0 0 -----

020 475812 4999140 BLANK 2 0 0 ----- 0 0 -----

021 475812 4999140 ----- 2 0 0 ----- 0 0 -----

022 475455 4998797 ----- 2 0 0 ----- 0 0 -----

023 475459 4998787 ----- 2 0 0 ----- 0 0 -----

024 475492 4998767 ----- 2 0 0 ----- 0 0 -----

025 475516 4998772 ----- 2 0 0 ----- 0 0 -----

026 475530 4998774 ----- 2 0 0 ----- 0 0 -----

027 475591 4998738 ----- 2 0 0 ----- 0 0 -----

028 475611 4998764 ----- 2 0 0 ----- 0 0 -----

029 476098 4998751 ----- 2 0 0 ----- 0 0 -----

030 475658 4998831 ----- 2 0 0 ----- 0 0 ----- about 100 gulls

031 475671 4998814 ----- 2 0 0 ----- 0 0 ----- about 100 gulls

032 475595 4998879 BLANK 1 0 0 ----- 0 0 -----

033 475595 4998879 ----- 2 0 0 ----- 0 0 -----

034 475586 4998840 ----- 4 0 0 ----- 0 0 -----

035 475719 4998775 ----- 2 0 0 ----- 0 0 -----

036 475788 4998733 ----- 2 0 0 ----- 0 0 -----

037 475851 4998700 ----- 2 0 0 ----- 0 0 -----

038 475963 4998657 ----- 2 0 0 ----- 0 0 -----

039 475665 4998568 ----- 2 0 0 ----- 0 0 -----

040 475683 4998639 ----- 2 0 0 ----- 0 0 -----

041 476111 4998731 ----- 4 0 0 ----- 0 0 -----

042 476120 4998708 ----- 2 0 0 ----- 0 0 -----

043 476129 4998681 ----- 2 0 0 ----- 0 0 -----

044 476209 4998407 ----- 2 0 0 ----- 0 0 -----

045 476222 4998385 ----- 2 0 0 ----- 0 0 -----

046 476233 4998355 BLANK 1 0 0 ----- 0 0 -----

047 476233 4998324 ----- 2 0 0 ----- 0 0 -----

048 476244 4998324 ----- 2 0 0 ----- 0 0 -----

049 476263 4998215 ----- 6 0 0 ----- 0 0 -----

050 476274 4998188 ----- 2 0 0 ----- 0 0 -----

051 476276 4998162 ----- 2 0 0 ----- 0 0 -----

052 476282 4998145 ----- 6 0 0 ----- 0 0 -----

053 476296 4998112 ----- 6 0 0 ----- 0 0 -----

054 Not collected
Appendixes 1–3 23

Date collected: September 25, 2012

Location: Mississippi River below Lock and Dam 1

Silver Carp Bighead Carp

NOTES

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

ID Easting Northing Blank

No. of 

Filters

001 484088 4973332 ----- 2 1 0 ----- 0 0 -----

002 484063 4973334 ----- 2 0 0 ----- 0 0 -----

003 484027 4973295 ----- 2 0 0 ----- 0 0 ----- gull flew over

004 484137 4973159 ----- 2 1 0 ----- 0 0 -----

005 484221 4973235 ----- 2 0 0 ----- 0 0 ----- gull over island

006 484299 4973267 ----- 2 0 0 ----- 0 0 -----

007 484157 4973038 ----- 2 0 0 ----- 0 0 -----

008 484180 4972841 BLANK 1 0 0 ----- 0 0 -----

009 484180 4972841 ----- 2 0 0 ----- 0 0 ----- gull

010 484214 4972790 ----- 2 0 0 ----- 0 0 -----

011 484235 4972767 ----- 2 0 0 ----- 0 0 -----

012 484297 4972789 ----- 2 0 0 ----- 0 0 ----- eagle flying over

013 484397 4972694 ----- 2 0 0 ----- 0 0 -----

014 484388 4972626 ----- 2 0 0 ----- 0 0 -----

015 484794 4972340 ----- 2 0 0 ----- 0 0 -----

016 484819 4972306 ----- 2 0 0 ----- 0 0 -----

017 484916 4972291 ----- 2 0 0 ----- 0 0 -----

018 484968 4971827 ----- 2 0 0 ----- 0 0 -----

019 484931 4971818 ----- 2 0 0 ----- 0 0 -----

020 484875 4971816 ----- 2 0 0 ----- 0 0 -----

021 485039 4971421 ----- 4 1 0 ----- 0 0 -----

022 485064 4971364 ----- 2 0 0 ----- 0 0 -----

023 485054 4971332 ----- 2 0 0 ----- 0 0 -----

024 485138 4971285 ----- 2 0 0 ----- 0 0 -----

025 485306 4971223 ----- 2 1 0 ----- 0 0 -----

026 485319 4971261 ----- 2 0 0 ----- 0 0 -----

027 485327 4971300 ----- 2 0 0 ----- 0 0 -----

028 485691 4971250 ----- 2 0 0 ----- 0 0 -----

029 485677 4971219 ----- 2 1 0 ----- 0 0 -----

030 485647 4971190 ----- 2 0 0 ----- 0 0 -----

031 485745 4971181 ----- 2 0 0 ----- 0 0 -----

032 485785 4971168 ----- 2 0 0 ----- 0 0 -----

033 485976 4971124 ----- 2 0 0 ----- 0 0 -----

034 485983 4971047 ----- 2 0 0 ----- 0 0 -----

035 485966 4970950 ----- 2 0 0 ----- 0 0 -----

036 485957 4970833 BLANK 1 0 0 ----- 0 0 -----

037 485957 4970833 ----- 2 0 0 ----- 0 0 -----

038 485973 4970837 ----- 2 0 0 ----- 0 0 -----

039 486032 4971221 ----- 2 0 0 ----- 0 0 -----

040 486225 4971124 ----- 2 0 0 ----- 0 0 -----

041 486409 4971214 ----- 2 4 0 ----- 1 0

042 486453 4971230 ----- 2 0 0 ----- 0 0 -----

043 486456 4971284 ----- 4 0 0 ----- 0 0 -----

044 486454 4971243 ----- 2 0 0 ----- 0 0 -----

045 486717 4971439 ----- 2 0 0 ----- 0 0 -----

046 486812 4971400 ----- 2 2 0 ----- 0 0 -----

047 486954 4971170 ----- 2 4 0 ----- 0 0 -----

048 487216 4971287 ----- 2 0 0 ----- 0 0 -----

049 487664 4971517 BLANK 1 0 0 ----- 0 0 -----

050 487664 4971517 ----- 2 3 0 ----- 0 0 -----

051 488189 4971550 ----- 2 2 0 ----- 0 0 -----

052 487851 4971625 ----- 2 4 0 ----- 0 0 -----

053 487416 4971526 ----- 2 2 0 ----- 0 0 -----

054 ----- ----- Filter + 3 8 ----- ----- ----- ----- -----
24 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Date collected: October 1, 2012

Location: Square Lake

Silver Carp Bighead Carp

NOTES

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

ID Easting Northing Blank

No. of 

Filters

001 0515976 4999913 ----- 2 0 0 ----- 0 0 -----

002 0515846 4999852 ----- 1 0 0 ----- 0 0 -----

003 0515792 4999889 ----- 1 0 0 ----- 0 0 -----

004 0515749 4999924 ----- 1 1 0 ----- 0 0 -----

005 0515697 4999971 ----- 2 0 0 ----- 0 0 -----

006 0515644 4999999 ----- 1 0 0 ----- 0 0 -----

007 0515580 5000019 ----- 1 0 0 ----- 0 0 -----

008 0515544 5000039 ----- 1 0 0 ----- 0 0 -----

009 0515463 5000064 ----- 1 0 0 ----- 0 0 -----

010 0515416 5000084 ----- 2 0 0 ----- 0 0 -----

011 0515350 5000094 ----- 1 0 0 ----- 0 0 -----

012 0515295 5000088 ----- 1 0 0 ----- 0 0 -----

013 0515243 5000108 ----- 1 1 0 ----- 0 0 -----

014 0515168 5000097 ----- 1 1 0 ----- 0 0 -----

015 0515103 5000123 ----- 1 1 0 ----- 0 0 -----

016 0515030 5000149 ----- 1 0 0 ----- 0 0 -----

017 0514959 5000181 BLANK 1 0 0 ----- 0 0 -----

018 0514959 5000181 ----- 1 0 0 ----- 0 0 -----

019 0514881 5000277 ----- 1 0 0 ----- 0 0 -----

020 0514760 5000410 ----- 1 0 0 ----- 0 0 -----

021 0514769 5000541 ----- 1 0 0 ----- 0 0 -----

022 0514811 5000655 ----- 1 0 0 ----- 0 0 -----

023 0514839 5000715 ----- 1 0 0 ----- 0 0 -----

024 0514894 5000779 ----- 1 0 0 ----- 0 0 -----

025 0514965 5000834 ----- 1 0 0 ----- 0 0 -----

026 0515117 5000910 ----- 1 0 0 ----- 0 0 -----

027 0515208 5000909 ----- 1 0 0 ----- 0 0 -----

028 0515314 5000881 BLANK 1 0 0 ----- 0 0 -----

029 0515314 5000881 ----- 1 0 0 ----- 0 0 -----

030 0515428 5000812 ----- 2 0 0 ----- 0 0 -----

031 0515491 5000743 ----- 1 0 0 ----- 0 0 ----- kingfisher

032 0515569 5000636 ----- 2 0 0 ----- 0 0 -----

033 0515631 5000539 ----- 1 0 0 ----- 0 0 -----

034 0515679 5000443 ----- 1 0 0 ----- 0 0 -----

035 0515739 5000402 ----- 1 0 0 ----- 0 0 -----

036 0515827 5000373 ----- 1 1 0 ----- 0 0 -----

037 0515910 5000321 ----- 1 0 0 ----- 0 0 -----

038 0516003 5000325 ----- 1 0 0 ----- 0 0 -----

039 0516100 5000221 ----- 1 0 0 ----- 0 0 -----

040 0516076 5000066 ----- 1 0 0 ----- 0 0 -----

041 0515968 4999975 ----- 1 0 0 ----- 0 0 -----

042 0515912 5000012 ----- 1 2 0 ----- 0 0 -----

043 0515852 5000049 ----- 1 0 0 ----- 0 0 -----

044 0515749 5000099 ----- 1 1 0 ----- 0 0 -----

045 0515650 5000149 BLANK 1 0 0 ----- 0 0 -----

046 0515650 5000149 ----- 1 2 0 ----- 0 0 -----

047 0515581 5000175 ----- 1 3 0 ----- 0 0 -----

048 0515517 5000211 ----- 1 0 0 ----- 0 0 -----

049 0515441 5000248 ----- 1 0 0 ----- 0 0 -----

050 0515325 5000315 ----- 1 3 0 ----- 0 0 -----

051 0515210 5000368 ----- 1 0 0 ----- 0 0 -----

052 0515084 5000416 ----- 1 2 1 NO 0 0 -----

053 0514963 5000461 ----- 1 0 0 ----- 0 0 -----

054 ----- ----- Filter + 1 8 ----- ----- ----- ----- -----
Appendixes 1–3 25

Date collected: October 2, 2012

Location: Lake Riley

Silver Carp Bighead Carp

NOTES

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

ID Easting Northing Blank

No. of 

Filters

001 0459498 4964912 ----- 2 0 0 ----- 0 0 ----- hawk

002 0459462 4965020 ----- 2 0 0 ----- 0 0 -----

003 0459425 4965106 ----- 2 0 0 ----- 0 0 -----

004 0459310 4965196 ----- 2 0 0 ----- 0 0 -----

005 0459123 4965208 ----- 2 0 0 ----- 0 0 -----

006 0459049 4965131 ----- 2 0 0 ----- 0 0 -----

007 0458946 4965102 ----- 2 0 0 ----- 0 0 -----

008 0458865 4965154 ----- 2 0 0 ----- 0 0 -----

009 0458821 4965248 ----- 2 0 0 ----- 0 0 -----

010 0458798 4965313 ----- 2 0 0 ----- 0 0 -----

011 0458797 4965326 ----- 2 0 0 ----- 0 0 -----

012 0458791 4965417 ----- 2 0 0 ----- 0 0 -----

013 0458600 4965544 ----- 2 0 0 ----- 0 0 -----

014 0458445 4965394 BLANK 1 0 0 ----- 0 0 -----

015 0458445 4965394 ----- 2 0 0 ----- 0 0 -----

016 0458410 4965256 ----- 2 0 0 ----- 0 0 -----

017 0458384 4965125 ----- 2 1 0 ----- 0 0 -----

018 0458362 4965007 ----- 2 0 0 ----- 0 0 -----

019 0458316 4964905 ----- 2 0 0 ----- 0 0 -----

020 0458236 4964802 ----- 2 0 0 ----- 0 0 -----

021 0458163 4964762 ----- 2 0 0 ----- 0 0 -----

022 0458085 4964774 ----- 2 0 0 ----- 0 0 -----

023 0457995 4964825 ----- 2 0 0 ----- 0 0 -----

024 0457912 4964780 ----- 2 1 0 ----- 0 0 -----

025 0457892 4964679 ----- 2 2 0 ----- 0 0 -----

026 0457914 4964613 ----- 2 0 0 ----- 0 0 -----

027 0458034 4964596 ----- 2 0 0 ----- 0 0 -----

028 0458190 4964504 ----- 2 0 0 ----- 0 0 -----

029 0458313 4964428 BLANK 1 0 0 ----- 0 0 -----

030 0458313 4964428 ----- 2 0 0 ----- 0 0 -----

031 0458450 4964392 ----- 2 0 0 ----- 0 0 -----

032 0458667 4964407 ----- 2 1 0 ----- 0 0 ----- loon

033 0458835 4964432 ----- 2 1 0 ----- 0 0 -----

034 0459112 4964395 ----- 2 0 0 ----- 0 0 -----

035 0459264 4964498 ----- 2 0 0 ----- 0 0 -----

036 0459362 4964490 ----- 2 0 0 ----- 0 0 -----

037 0459411 4964563 ----- 1 0 0 ----- 0 0 -----

038 0459434 4964623 ----- 2 0 0 ----- 0 0 -----

039 0459477 4964725 ----- 2 0 0 ----- 0 0 -----

040 0459509 4964765 ----- 2 0 0 ----- 0 0 -----

041 0459431 4964768 ----- 2 0 0 ----- 0 0 -----

042 0459346 4964761 ----- 2 0 0 ----- 0 0 -----

043 0459217 4964766 ----- 2 1 0 ----- 0 0 -----

044 0459094 4964755 ----- 2 0 0 ----- 0 0 -----

045 0458917 4964752 ----- 2 0 0 ----- 0 0 -----

046 0458797 4964748 ----- 2 0 0 ----- 0 0 -----

047 0458678 4964724 ----- 2 0 0 ----- 0 0 -----

048 0458570 4964717 BLANK 1 0 0 ----- 0 0 -----

049 0458570 4964717 ----- 2 0 0 ----- 0 0 -----

050 0458482 4964688 ----- 2 0 0 ----- 0 0 -----

051 0458394 4964694 ----- 2 0 0 ----- 0 0 -----

052 0458276 4964690 ----- 2 0 0 ----- 0 0 -----

053 0458179 4964675 ----- 2 0 0 ----- 0 0 -----

054 ------- ------- Filter + 1 8 ----- ----- ----- ----- -----
26 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Date collected: October 16, 2012

Location: Mississippi River below Lock and Dam 19

Silver Carp Bighead Carp

NOTES

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

Presumptive 

PCR-Positives

Determinative 

PCR-Positives

Confirmed

ID Easting Northing Blank

No. of 

Filters

001 0638068 4472597 ----- 4 8 8 YES 0 0 -----

002 0638166 4472670 ----- 4 7 7 YES 0 0 -----

003 0638238 4472724 ----- 4 8 6 YES 0 0 -----

004 0638262 4472847 ----- 4 1 0 ----- 0 0 -----

005 0638246 4472849 ----- 4 2 4 YES 0 0 -----

006 0638228 4472776 ----- 4 8 8 YES 0 0 -----

007 0638456 4472586 ----- 4 8 7 YES 0 0 -----

008 0638497 4472605 ----- 4 7 7 YES 0 0 -----

009 0638550 4472584 ----- 4 7 6 YES 0 0 -----

010 0638614 4472479 ----- 4 7 5 YES 0 0 -----

011 0638694 4472412 ----- 3 0 0 ----- 0 0 -----

012 0638253 4472149 ----- 4 1 0 YES 0 0 -----

013 0638162 4472180 BLANK 4 0 0 ----- 0 0 -----

014 0638162 4472180 ----- 4 0 0 ----- 0 0 -----

015 0638004 4472230 ----- 4 8 4 YES 0 0 -----

016 0637815 4472262 ----- 4 8 8 YES 0 0 -----

017 0637775 4472195 ----- 4 8 8 YES 0 0 -----

018 0637524 4472063 ----- 4 0 0 ----- 0 0 -----

019 0637457 4472007 ----- 4 3 5 YES 0 0 -----

020 0637235 4471905 ----- 4 4 7 YES 0 0 -----

021 0637223 4471879 ----- 4 0 0 ----- 0 0 -----

022 0637118 4471687 ----- 4 4 4 YES 0 0 -----

023 0637091 4471562 ----- 4 3 2 YES 0 0 -----

024 0637191 4471514 ----- 4 0 0 ----- 0 0 -----

025 0637257 4471443 ----- 4 6 ----- ----- 0 0 -----

026 0637270 4471388 ----- 4 4 ----- ----- 0 0 -----

027 0637244 4471312 ----- 4 4 ----- ----- 0 0 -----

028 0637243 4471189 ----- 4 0 0 ----- 0 0 -----

029 0637343 4471235 BLANK 1 0 0 ----- 0 0 -----

030 0637343 4471235 ----- 4 0 0 ----- 0 0 -----

031 0637195 4471122 ----- 4 4 ----- ----- 0 0 -----

032 0637298 4471124 ----- 4 0 0 ----- 0 0 -----

033 0637070 4471109 ----- 4 8 ----- ----- 0 0 -----

034 0636931 4471140 ----- 2 8 ----- ----- 0 0 -----

035 0636924 4471093 ----- 4 1 ----- ----- 0 0 -----

036 0636705 4470983 ----- 4 0 0 ----- 0 0 -----

037 0636574 4470980 ----- 4 0 0 ----- 0 0 -----

038 0636463 4471032 ----- 4 4 ----- ----- 0 0 -----

039 0636405 4471069 ----- 4 0 0 ----- 0 0 -----

040 0636338 4471112 ----- 4 0 0 ----- 0 0 -----

041 0636268 4471191 ----- 4 0 0 ----- 0 0 -----

042 0636186 4471315 ----- 4 8 ----- ----- 0 0 -----

043 0635730 4471479 ----- 4 8 ----- ----- 0 0 -----

044 0635465 4471551 ----- 4 8 ----- ----- 0 0 -----

045 0635529 4471588 ----- 4 7 ----- ----- 0 0 -----

046 0635633 4471597 ----- 4 8 ----- ----- 0 0 -----

047 0635709 4471601 ----- 4 8 ----- ----- 0 0 -----

048 0635905 4471591 BLANK 1 0 0 ----- 0 0 -----

049 0635905 4471591 ----- 4 0 0 ----- 0 0 -----

050 0636239 4471614 ----- 4 8 ----- ----- 0 0 -----

051 0636380 4471568 ----- 4 0 0 ----- 0 0 -----

052 0637025 4471838 ----- 4 0 0 ----- 0 0 -----

053 0636533 4471724 ----- 4 7 ----- ----- 0 0 -----

054 Not collected
Appendix 2. Representative electrophoresis gels for the detection of the DNA of silver carp in samples taken 

from each site. Gels used for the detection of bighead carp were not included because bighead carp DNA was not 

detected. Representative gels for presumptive and determinative PCR-positives are included.

Appendixes 1–3 27

Appendix 2A. Representative electrophoresis gels demonstrating the detection of the DNA of silver carp 

in samples taken from the Mississippi River below Lock and Dam 19. Lanes considered presumptive PCRpositive are marked with “*”; the positive extraction and positive PCR control lanes are marked with a white 

“+”. 
28 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Appendix 2B. Representative electrophoresis gels demonstrating the detection of the DNA of 

silver carp in samples taken from Square Lake. Lanes considered presumptive PCR-positive are 

marked with “*”; the positive extraction and positive PCR control lanes are marked with a white 

“+”. 
Appendixes 1–3 29

Appendix 2C. Representative electrophoresis gels demonstrating the detection of the DNA of 

silver carp in samples taken from Lake Riley. Lanes considered presumptive PCR-positive are 

marked with “*”; the positive extraction and positive PCR control lanes are marked with a white 

“+”.
30 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Appendix 2D. Representative electrophoresis gels demonstrating the detection of the DNA of silver 

carp in samples taken from the Mississippi River above the Coon Rapids Dam. Lanes considered 

presumptive PCR-positive are marked with “*”; the positive extraction and positive PCR control lanes 

are marked with a white “+”. 
Appendixes 1–3 31

Appendix 2E. Representative electrophoresis gels demonstrating the detection of the DNA of silver 

carp in samples taken from the Mississippi River below the Coon Rapids Dam. Lanes considered 

presumptive PCR-positive are marked with “*”; the positive extraction and positive PCR control lanes are 

marked with a white “+”. 
32 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Appendix 2F. Representative electrophoresis gels demonstrating the detection of the DNA of 

silver carp in samples taken from the Mississippi River below Lock and Dam 1. Lanes considered 

presumptive PCR-positive are marked with “*”; the positive extraction and positive PCR control 

lanes are marked with a white “+”.
Appendixes 1–3 33

Appendix 2G. Representative electrophoresis gels demonstrating the detection of the DNA 

of silver carp in samples taken from the St. Croix River above the St. Croix Falls Dam. Lanes 

considered presumptive PCR-positive are marked with “*”; the positive extraction and positive 

PCR control lanes are marked with a white “+”. 
34 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Appendix 2H. Representative electrophoresis gels demonstrating the detection of the DNA of silver carp 

in samples taken from the St. Croix River below the St. Croix Falls Dam. Lanes considered presumptive 

PCR-positive are marked with “*”; the positive extraction and positive PCR control lanes are marked with a 

white “+”.
Appendixes 1–3 35

Appendix 2I. Representative electrophoresis gels of samples considered to be presumptive PCR positive from 

Square Lake and Lake Riley. Lanes considered determinative PCR-positives are marked with “**”; the positive 

PCR control lanes are marked “++”.
36 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Appendix 2J. Representative electrophoresis gels for samples considered to be presumptive PCR positive from 

Lake Riley and from the Mississippi River below Lock and Dam 19. Lanes considered determinative PCR-positive 

are marked with “**”; the positive PCR control lanes are marked “++”.
Appendixes 1–3 37

Appendix 2K. Representative electrophoresis gels for samples considered presumptive PCR positive from above 

the St. Croix Falls Dam, above the Coon Rapids Dam, below the St. Croix Falls Dam, below the Coon Rapids Dam 

and below Lock and Dam 1. Lanes considered determinative PCR-positive are marked with “**”; the positive PCR 

control lanes are marked “++”.
38 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Appendix 3. Sequences of determinative PCR-positive environmental samples amplified with the silver carp marker. 

The percent match of each sequence to published silver carp DNA sequences in GenBank (http://www.ncbi.nlm.nih.

gov/genbank/ ) is included following the sequence. A sample was determined “confirmed positive” only when both 

the forward and reverse sequences of that sample were greater than 95% similar to published silver carp sequences. 

Lock and Dam 19 - 2012 1016 001 – CONFIRMED POSITIVE

Forward

TGGGGGTCTCAATTTTTCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCACAATGAACTATTACTTGCATTTGGCTTAACTTAAATCCAATGGTTGATCCAATGGCACGGCCTCCCAAAACAGAAGGGTTAAACCAAAAAACCTGAACTTATCCCTTCCTAATCCTATTAAAATTAAAAAAACCACCCCCCTAAAAGAAAAAATTACTTTATTTTAAACATCTTTTTGTAAACCCTCCCACCGCGCTTTGTAAATTTCAATAACATATTCTTTTTTTTTATATTTACAGCACATCCGTGCGACGTCAAAGAAATGAAACCACACTACAGATGAAATGATAGAAAACTTTAGA

96% match to silver carp on >150 nucleotide portion of sequence

Reverse 

GGGGACCCCACGATTATTGTCCCTGATCTTATCATGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGGACCAATTATAGTGGAACACCTCTTTTCTCAGGATTTTCTTTATTTTTATGGTTAAGGGAGGGGCCCCCGCCCCACAAAACGGGAAGGGAACACCCCCGGTCCTGGCCCCATACCTCCTCAACCACTCAAAATTACGTGCACCCCCCCCGAAAAAAGAACTTTTTCTTTTTTTTTTTTTTTTTTTACCAGCCCCCCGCCTTTTTGTTTTTTTAAATTCCTTTTCTCTTTTTTTTTTTTCTCAGAACCGCGGAGACGCGTGAAGCCAATGAACACTACCTACAGAGAAGATGATAGGTTACTC

98% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 002– CONFIRMED POSITIVE

Forward 

GGGGCTCTAAACATCTTCCTTTGCAATATTAACTTCTATTTAACTTAACTATATTAATTGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCACACACCCCCGGGCCCTGCTGGGGAGAAACAAAAAAAAAACACAACACCCCACCCACAGTACGGACCGGCCTTTTATAAGGGTCTTAT

95% match to silver carp on >150 nucleotide portion of sequence

Reverse 

GGGGCCCCGCACGCATTATTTGTCCCTGATCTTATCATGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGACAACCAATTATAGTGGAACACCTCTTTTCTCAGGACCCCCGTTCCCCCCCAGCTATATCCACAAAATCCACCGCCACTCCAGCCCGGTGCTTCCCCCCTTCCCATCTAGGTCTATT

96% match to silver carp on >150 nucleotide portion of sequence
Appendixes 1–3 39

Lock and Dam 19 - 2012 1016 003– CONFIRMED POSITIVE

Forward 

TGTTGGTTCTCAAATATTTCCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCATTTTTCTTTTTTCTTTTGGTTTATGCAAGGGCCTGGTCCCCCAAAACGGGAAGGGTAAATCCCCGGACCTGACCCTATCCCTTCATCAACCACTCCTAATTACAATCTCCACCCCAAAAACAAAACTTTTCCTTTTTTTAAATTTTTCTTTTTAAGTGGTCCTTCGCTTTTGATTTTTTAATACCGTTTCTCTCCTTTTCATTTCAGCGTTACCGGCGTGCTTTGTGAAAGAC

99% match to silver carp on >150 nucleotide portion of sequence

Reverse 

TGGACCCCACGATTATTGTCCCTGATCTTATCATGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTCTCAGGGTTTTTCTTTTTTTTTTTGTTTTATGGAATGGCCCGCCCAAAAAAAACGGAAAGGGAAAAATACCCATTCTGGTCCCTTACTTTCACTCCACTTATAATTAACTGTATCGCCACGAAAAAAAAATTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAACCCCCCGGGGGCGTTTTGTTATTTAATATAATATTTCTCCCTTTTATATTTATCAAACCCGGTGTGAGATGTGA

98% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 005 – CONFIRMED POSITIVE

Forward 

GTTGGGTTCAATATTTCCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCACACCCCCCGTGCCTGGCTGGGAGAACCAAAAAAAAAAACCCAACCCCCACCCCCGGGGGGGGCCCCCTTATAAACAAGGGTTATA

99% match to silver carp on >150 nucleotide portion of sequence

Reverse 

CCCCCACGGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTCTCAGGGCCCCCGTCCCTCCTGGGAGAGACAAGAAAAAAACGGACACCCCATCCGGGTAGGGACCTGCCATTAGTCGGGT

98% match to silver carp on >150 nucleotide portion of sequence
40 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Lock and Dam 19 - 2012 1016 006 – CONFIRMED POSITIVE

Forward 

TGGGGTTCATTTTCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCAACCCGGGTCCCGCATGGGGGGACAACAAAAAACTGGACACCCCCAACCGGGGGGGGCCCCCCTTCCCTGCTGAGAAA

99% match to silver carp on >150 nucleotide portion of sequence

Reverse 

GGACCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTCTCAGG

98% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 007 – CONFIRMED POSITIVE

Forward 

TGGGGTCTAATATTTCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCA

98% match to silver carp on >150 nucleotide portion of sequence

Reverse 

CCCCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTCTCAGG

98% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 008 – CONFIRMED POSITIVE

Forward

GGGGGTCCATTTTCCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCA

99% match to silver carp on >150 nucleotide portion of sequence

Reverse

CCCCCCGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTCTCAGG

97% match to silver carp on >150 nucleotide portion of sequence
Appendixes 1–3 41

Lock and Dam 19 - 2012 1016 009 – CONFIRMED POSITIVE

Forward

GGGGGTCTCAATTTTCCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCA

97% match to silver carp on >150 nucleotide portion of sequence

Reverse

GGGACCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACAAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTCTCAGG

97% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 010 – CONFIRMED POSITIVE

Forward

GTGGGGTCCAATTTTTCCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCCTTTGGCA

97% match to silver carp on >150 nucleotide portion of sequence

Reverse

GGCCCCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACAAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGGACCAATTATAGTGGAACACCTCTTTTCTCAGGCC

97% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 015 – CONFIRMED POSITIVE

Forward

GGGGGTCCTCATATTTCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCCCATAATGAACTATTACTTGCATTTGGCACCC

96% match to silver carp on >150 nucleotide portion of sequence

Reverse

GGGACCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACAAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAG

98% match to silver carp on >150 nucleotide portion of sequence
42 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Lock and Dam 19 - 2012 1016 016 – CONFIRMED POSITIVE

Forward

GCTGGTCTCATATTTCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCA

99% match to silver carp on >150 nucleotide portion of sequence

Reverse

GCCCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTCTCAGGA

98% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 017 – CONFIRMED POSITIVE

Forward

GTTGGGTCTCATTTTCCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGG

95% match to silver carp on >150 nucleotide portion of sequence

Reverse

GGGCCGCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACAAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTCTCAGG

97% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 019 – CONFIRMED POSITIVE

Forward

GCGGGGTCCAAAATTTCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCACCCCCGGGCCCCCCCGGCTATAACACCAAAATCCACGGCCACCCCCGGCCCGGTGCTTCCCCCCTTCCCTTCTGAAGATTAACC

97% match to silver carp on >150 nucleotide portion of sequence

Reverse

GGGGCCCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTCTCAGGG

98% match to silver carp on >150 nucleotide portion of sequence
Appendixes 1–3 43

Lock and Dam 19 - 2012 1016 020 – CONFIRMED POSITIVE

Forward

GTTTGGTCTCATATTTCCTTGAATATTAACTTCTATTTAACTTAACTATATAAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCAACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGCTGCCTAATGACTGAGCTAACTCATATTAATTGTTTTCTGTTTATTGTCTGCTTTTCCCCCGGAAACTTGTCTTGTCACCTGCATTAATGAATCGGCCAACCTGCGGGGACACGCGG

99% match to silver carp on >150 nucleotide portion of sequence

Reverse

GGGTCCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACAAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGA

98% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 022 – CONFIRMED POSITIVE

Forward

GCGGGTCTAATTTTCCTTGAATATTAACTTCTATTTAACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGC

99% match to silver carp on >150 nucleotide portion of sequence

Reverse

GGGGCCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACAAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGAACCAATTATAGTGGAACACCTCTTTTTTCAGG

97% match to silver carp on >150 nucleotide portion of sequence

Lock and Dam 19 - 2012 1016 023 – CONFIRMED POSITIVE

Forward

TTTGGTCTAATTTTCCTTGAATATTAACTTCTATTTACTTAACTATATTAATGTAGTAAGAAACCACCTACTGGTTTATATTAAGGTATTCTATTCATGATAAGATCAGGGACAATAATCGTGGGGGTGGCGCAGAATGAACTATTACTTGCATTTGGCA

99% match to silver carp on >150 nucleotide portion of sequence

Reverse

GTGACCCACGATTATTGTCCTGATCTTATCATGAATAGAATACCTTAATATAAACAAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGAGGACCAATTATAGTGGAACACCTCTTTTCTCAGGCAC

97% match to silver carp on >150 nucleotide portion of sequence
44 Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River

Square Lake - 2012 1001 052 – CONFIRMED NEGATIVE

Forward

CGGGGCCCAAAATCCTCCTTGGATTATCAACTTCTATTTAACTTGACTATATTAATGGGTAATAACCGGGGACTGGTTTATAGAAAGGTTTCTCCTCATGAGGAGATCCGGGAACGCCGCGTGGGGGTGGCGGTTATTGAACTCTTCCTTGGCCTGTCACACCCCCGGCCCCGCTGGGGGGGCCAGCGAATAAAC

77% match to silver carp on 135 nucleotide portion of sequence

Reverse

GCCCCCCACGATTATTGTCCTTGATCTTATCTGAATAGAATACCTTAATATAAACCAGTAGGTGGTTTCTTACTACATTAATATAGTTAAGTTAAATAGAAGTTAATATTTCAAGGAAATATTTGACAACCAATTATAGTGGAACACCTCTTTTCTCTGGATC

96% match to silver carp on >150 nucleotide portion of sequence

St. Croix, above dam - 2012 0917 016 – CONFIRMED NEGATIVE

Forward

CGGCCCCCTTTTTTTCGCTTGTTTTTTCCCTCTGTTTGAGCGTCGGAATCTCTTGAGCCTATTCTCTTACAAAGCATCTTATAGGTCTTCGTTAATTTTCGGCTTGTTTTCAATGACCCATTACTAGGATTTCGCACGTTTACTATCCTCCCCCCATGTTTCTGCCCACCCCCGTTT

0% match to silver carp

Reverse

TGGCCCGAACATTTATTGAATGACCCACGAGATGCTTTATTAAGAGAATATGCTCAAGAAATAGCGAGGGTCAAACAGATGCTGGAACAACAAGAAATAGGAGAGCCTATTATAGTGGAACACCTCTTTTCTCAGGGGTT

87% match to silver carp on only a 44 nucleotide portion of sequence

St. Croix, above dam - 2012 0917 043 – CONFIRMED NEGATIVE

Forward

CGGGGGCCTCACTGGTACCTGGGTTTTTTAACTCCTCTTGACTGACCCTTTTGTGAGAAAGAGCCCCTACTCCATATATTAAGGCTCTCTCCATGAGATGATCAGGGACAACAGTCATGGGCGGGCGTTTAATGAACTCCCCCTAGCTTTTCCAAACCCCCGGGCCCGGCTGGCAAGTACAAAATTAAAACCC

78% match to silver carp on only a 63 nucleotide portion of sequence

Reverse

TCCCCCCCCCCAAATTATTTTTCCCTATTTTTATCTGAATAAATTACCTTAATATAACCCATTAGGGGGTTTCTTACTATTTTAATATACGTAACTTAAATAGAATTTATTATTTCAGGGAAATTTTGATTCACGAATTTAATTGAAATCCTTCTTTTCTCAC

80% match to silver carp on 154 nucleotide portion of sequence

Amberg and others—Detection of Environmental DNA of Bigheaded Carps in Samples from the St. Croix River and in the Mississippi River—Open-File Report 2013–1080