Prepared in cooperation with Wisconsin Cooperative Fishery Research Unit, 

Molecular Conservation Genetics Laboratory, College of Natural Resources, 

University of Wisconsin-Stevens Point

Validation of eDNA Markers for New Zealand Mudsnail

Surveillance and Initial eDNA Monitoring at 

Mississippi River Basin Sites 

By Christopher M. Merkes, Keith N. Turnquist, Christopher B. Rees, and Jon J. Amberg

Open-File Report 2015-1037

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

SALLY JEWELL, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2015

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Suggested citation:

Merkes, C.M, Turnquist, K.N, Rees, C.B, and Amberg, J.J, 2015, Validation of eDNA markers for New 

Zealand mudsnail surveillance and initial eDNA monitoring at Mississippi River Basin sites: U.S. 

Geological Survey Open-File Report 2015–1037, 9 p., http://dx.doi.org/10.3133/ofr20151037. 

ISSN 2331-1258 (online)
iii

Acknowledgments

The authors would like to acknowledge John C. Nelson for generation of the map used in 

figure 1. Maureen Ferry coordinated the preparation for the study. Jean Unmuth, Michael Sorge, 

Kim Bogenschutz, Jason Euchner, James Amrhein, Rachel Peacher, David Wyffels, Matt 

O’Hara, Blake Bushman, Blake Ruebush, Kurt Rasmussen, Jacob Schweitzer, Mark Hazuga, 

Hnue Yang, Dave Farrow, and Amanda Perdzock collected the monitoring samples. This work 

was funded by the Mississippi Interstate Cooperative Resource Association and the Wisconsin 

Department of Natural Resources.

Contents

Abstract ...................................................................................................................................................................... 1

Introduction................................................................................................................................................................. 2

Materials and Methods ............................................................................................................................................... 2

Marker Validation Sample Preparation.................................................................................................................... 2

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

DNA Extraction ....................................................................................................................................................... 4

Quantitative Polymerase Chain Reaction (qPCR) Analysis .................................................................................... 4

DNA Sequencing .................................................................................................................................................... 5

Data Analysis.......................................................................................................................................................... 5

Results........................................................................................................................................................................ 5

References ................................................................................................................................................................. 8

Appendix Sample collection data sheet. .................................................................................................................. 9

Figures

1. Map showing the locations of sampling sites................................................................................................. 7

Tables

1. List of New Zealand mudsnail eDNA monitoring sites. .................................................................................. 3

2. Temperature profiles for quantitative polymerase chain reaction assays. ..................................................... 4

3. Oligonucleotide sequences used for quantitative polymerase chain reaction assays.................................... 4

4. Upper Midwest Environmental Sciences Center assay validation results...................................................... 6

5. Molecular Conservation Genetics Laboratory assay validation results.......................................................... 6

6. Sequencing results. ....................................................................................................................................... 6

7. Monitoring results. ......................................................................................................................................... 8

v
vi

Abbreviations

BLAST Basic Local Alignment Search Tool

coi Cytochrome C Oxidase Subunit I gene 

cytb Cytochrome B gene

eDNA environmental DNA 

g standard gravity

M molar

μM micromolar

μL microliter

MCGL Molecular Conservation Genetics Laboratory, College of Natural Resources, 

University of Wisconsin-Stevens Point 

NCBI National Center for Biotechnology Information 

NZMS New Zealand mudsnail 

qPCR Quantitative Polymerase Chain Reaction

UMESC Upper Midwest Environmental Sciences Center

USGS U.S. Geological Survey 

Conversion Factors

Inch/Pound to International System of Units

Multiply By To obtain

Length

foot (ft) 0.3048 meter (m)

yard (yd) 0.9144 meter (m)

Volume

barrel (bbl; petroleum, 1 barrel=42 gal) 0.1590 cubic meter (m3) 

ounce, fluid (fl. oz) 0.02957 liter (L) 

Mass

ounce, avoirdupois (oz) 28.35 gram (g) 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as °F = (1.8 × °C) + 32.

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as °C = (°F – 32) / 1.8.
Validation of eDNA Markers for New Zealand Mudsnail

Surveillance and Initial eDNA Monitoring at 

Mississippi River Basin Sites 

By Christopher M. Merkes1, Keith N. Turnquist2, Christopher B. Rees1, and Jon J. Amberg1 

1

U.S. Geological Survey

2

University of Wisconsin-Stevens Point 

Abstract

The performance of newly developed New Zealand mudsnail (Potamopyrgus 

antipodarum; NZMS) genetic markers for environmental (eDNA) analysis of water were 

compared across two laboratories. The genetic markers were tested in four quantitative 

polymerase chain reaction assays targeting two regions of the NZMS mitochondrial genome, 

specifically the cytochrome c oxidase subunit 1 (coi) and cytochrome b (cytb) genes. In a blind 

study, analysts tested each sample eight times with each assay. There were 10 expected-negative 

samples from the Black River in La Crosse, Wisconsin, 10 expected-positive samples from the 

Black Earth Creek in Black Earth, Wisconsin, and 10 known-positive samples from the Black 

River spiked with NZMS DNA. Previously extracted samples, kept at the Upper Midwest 

Environmental Sciences Center, were pooled by sample location and then equal quantities were 

distributed between the Upper Midwest Environmental Sciences Center and the Molecular 

Conservation Genetics Laboratory at the University of Wisconsin-Stevens Point for analysis. The 

assays tested were (1) the assay targeting cytb with a minor groove binder probe described by 

Goldberg and others (2013), (2) the cytb assay with a modified double-quenched probe, (3) an 

assay targeting coi with a double-quenched probe, and (4) a duplex reaction combining the 

modified cytb assay and the coi assay. Samples were considered positive for the presence of 

NZMS DNA when quantitative polymerase chain reaction amplification and probe signal was 

higher than the normalized threshold value above baseline fluorescence. For the duplex assay, 

samples were considered positive only when both probe signals were higher than the normalized 

threshold value above baseline fluorescence. Positive results were then confirmed by sequencing 

the products. 

All four assays detected the DNA of NZMS in all expected-positive and known-positive 

samples in both labs. The modified cytb assay, the coi assay, and the duplex assay all failed to 

detect the DNA of NZMS in all expected-negative samples in both labs. The cytb assay, as 

described by Goldberg and others (2013), failed to detect the DNA of NZMS in all expectednegative samples for the Molecular Conservation Genetics Laboratory, but some reactions 

resulted in positive detection in late cycles for 9 of the 10 expected-negative samples at the 

Upper Midwest Environmental Sciences Center. Amplicons for expected-negative samples with 

 
2

positive reactions were sent for sequencing, and none were confirmed as NZMS. Six amplicons 

failed to give readable sequences, and three gave sequences without similarity to any known 

sequence in GenBank. Amplicons from each assay for one representative positive sample were 

sequenced and identified as NZMS with greater than 99 percent identity.

The duplex assay was chosen as the most efficient assay and was used at the Upper 

Midwest Environmental Sciences Center to analyze triplicate samples from 29 streams in 

Wisconsin, 8 streams in Illinois, and 8 streams in Iowa. In order to verify results, additional 

triplicate samples were collected from two of the streams in Iowa and two of the streams in 

Wisconsin for analysis at the Molecular Conservation Genetics Laboratory. All samples at all 

sites were negative for NZMS DNA.

Introduction

Environmental DNA (eDNA) has been developed as an early detection and surveillance 

tool for New Zealand mudsnails (Potamopyrgus antipodarum; NZMS) in response to the 

growing invasion in the Great Lakes and western states (Goldberg and others, 2013). In October 

2013, NZMS specimens were identified in benthic macroinvertebrate samples collected from 

Black Earth Creek, Dane County, Wisconsin during 2011 and 2012. This was the first known 

discovery of NZMS in the Midwest outside of the Great Lakes Basin and represents a significant 

threat to State and regional water resources. Following this discovery, additional benthic and 

eDNA samples were collected from Black Earth Creek. Preliminary results indicated that eDNA 

may be more accurate than traditional benthic sampling in the detection of NZMS in areas with 

low population densities.

In addition to the genetic marker developed by Goldberg and others (2013) targeting the 

cytochrome b mitochondrial gene (cytb), the Molecular Conservation Genetics Laboratory 

(MCGL) at University of Wisconsin – Stevens Point (UWSP) developed another marker for 

NZMS detection targeting the cytochrome c oxidase subunit I mitochondrial gene (coi). New 

markers that have been rigorously tested or published by other labs should be further validated 

before implementation in any monitoring program. This additional validation should be 

completed in multiple labs to demonstrate that the new markers are robust and produce 

repeatable results with different personnel and across different platforms. 

A two-lab validation study was completed to compare the results of the newly developed 

markers following the procedures used to validate markers for eDNA surveillance of Asian carp 

(U.S. Geological Survey, U.S. Fish and Wildlife Service, U.S. Army Corps of Engineers, unpub. 

data, 2014). The validated markers were then used to test water samples collected from 45 sites 

in Wisconsin, Illinois, and Iowa for New Zealand mudsnail DNA.

Materials and Methods

Marker Validation Sample Preparation

Previously archived DNA extracts from Black River water samples collected in March, 

2013, in La Crosse, Wisconsin were used as the expected-negative samples. Previously archived 

DNA extracts from Black Earth Creek water samples collected at South Valley Road in 

September, 2014, in Black Earth, Wisconsin were used as the expected-positive samples. 

Extracted DNA from captured NZMS was quantified at UMESC using the NZMS-cytb assay. 

Copy numbers were determined in 3 replicate reactions at each of 3 steps in a 10-fold serial 
3

dilution of starting material (9 total reactions), quantified from calibration with 2 replicate 

standard curves. Starting concentration was back-calculated by linear regression of the 

determined copy numbers and dilution factors from the 9 test reactions. Known concentrations at 

2 levels (high = 10,000 copies/microliter (μL) and low = 100 copies/μL) were then spiked into 

expected-negative extracts from Black River water samples. Extracts were pooled by type, and 

an analyst not otherwise associated with the study aliquoted 75 μL samples for each lab, 

numbered at random and documented on a separate data sheet. Ten expected-negative samples, 

10 expected-positive samples, 5 incurred-high samples, and 5 incurred-low samples were 

provided for each lab. Sample identity was unknown to all individuals associated with the study 

until the qPCR results were compiled. 

Monitoring Sample Collection

Triplicate water samples were collected from 45 sites for monitoring (table 1). Measuring 

downstream from the road crossing at each site, survey flags were placed every 10 meters (m) on 

shore for 100 m. Starting at the flag furthest downstream, 50 milliliters (mL) of water was 

collected from each 10 m block from the water surface targeting stream edges of low flow or 

areas of surface scum accumulation. The 50 mL water samples from the site were pooled into a 

500 mL composite water sample that was then agitated before pipetting 15 mL subsamples into 

each of three 50 mL conical tubes containing 33 mL absolute ethanol and 1.5 mL of 3 molar

sodium acetate for sample preservation. Preserved samples from all 45 sites were delivered to 

UMESC for testing. At four sites, three additional subsamples were taken from the composite 

sample for verification at the MCGL, as noted on table 1. 

Waders were scrubbed with a brush, soaked in a 2 percent Virkon solution for 20 

minutes, sprayed with bleach, and rinsed with municipal water between each site. Data sheets for 

ancillary information about the sample collection and site descriptions were filled out for each 

sample (Appendix A). 

Table 1. List of New Zealand mudsnail eDNA monitoring sites.

[Ill., Illinois; Wis., Wisconsin] 

Site State Site State Site State

Bloody Run1 Iowa Sugar River at Colored Sands Ill. Namekagon River Wis.

French Creek Iowa Bear Creek Wis. Prairie River Wis.

North Bear Creek Iowa Big Green River Wis. Reads Creek Wis.

Paint Creek Iowa Big Spring Creek Wis. Rowan Creek Wis.

South Bear Creek Iowa Bishop Branch Wis. Rush River Wis.

Sny Magill Creek Iowa Camp Creek Wis. Spring Creek Wis.

Spring Branch Iowa Castle Rock Creek Wis. Story Creek1 Wis.

Waterloo Creek1 Iowa Dell Creek Wis. Sugar River at Frenchtown1 Wis.

Apple River at East Canyon Road Ill. Duncan Creek Wis. Timber Coulee Creek Wis.

Apple River at Townsend Ill. Gilbert Creek Wis. Trimbelle River Wis.

Fox River at Ayers Landing Ill. Gordon Creek at Spring Creek 

Road

Wis. Trout Creek Wis.

Fox River at Fox River Drive

Bridge

Ill. Gordon Creek at State Route 39 Wis. Turton Creek Wis.

Kishwaukee River Ill. Kinnickinnic River Wis. West Branch Sugar River Wis.

Pecatonica River Ill. Mt. Vernon Creek Wis. West Fork Kickapoo Wis.

Rock River Ill. North Fork Bad Axe River Wis. Willow Creek Wis.

1

Additional replicate samples collected for results verification at the Molecular Conservation Genetics Laboratory. 
4

DNA Extraction

Samples were centrifuged at 5,000 times standard gravity (g) for 30 minutes, and the 

supernatant was decanted. DNA in the remaining pellets was extracted using the commercially 

available gMax Mini Genomic DNA kit (IBI Scientific; Peosta, Iowa) following the 

manufacturer’s guidelines with a final elution volume of 100 μL. 

Quantitative Polymerase Chain Reaction (qPCR) Analysis

The presence of New Zealand mudsnail DNA was determined using quantitative 

polymerase chain reaction (qPCR) assays. All assays were run in eight replicate reactions using 

20 μL total volume with 1 μL of template DNA. All assays used 2x SensiFAST Probe No-ROX 

master mix (Bioline; Taunton, Mass.) to supply pH buffer, DNA polymerase, Mg2+ coenzyme, 

deoxynucleotide triphosphates, and PCR adjuvants at 1x proprietary final concentration. The 

NZMS-cytb assay used 0.4 micromolar (μM) final concentration of each primer and 0.2 μM final 

probe concentration. The NZMS-cytb-mod, NZMS-coi, and NZMS-cytb-coi assays used 0.2 μM 

final concentration of each primer and 0.4 μM final concentration of each probe. The 

temperature profiles used are listed in table 2, and the oligonucleotide sequences used are listed 

in table 3. Assays were run on a Mastercycler Realplex 2 (EppendorfNA; Hauppage, N.Y.) at the 

Upper Midwest Environmental Sciences Center and on a MX3005P qPCR system (Stratagene; 

La Jolla, Calif.) at the Molecular Conservation Genetics Laboratory. 

Table 2. Temperature profiles for quantitative polymerase chain reaction assays. 

[NZMS, New Zealand mudsnail; °C, degrees Celsius; sec., seconds; min., minutes]

Step NZMS-cytb assay NZMS-cytb-mod, NZMS-coi, 

NZMS-cytb-coi assays

Initial Denaturation 95 °C – 2 min. 95 °C – 2 min.

Amplification and 

Quantification

50 cycles of: 55 cycles of:

94 °C – 60 sec. 95 °C – 60 sec.

60 °C – 60 sec. 52 °C – 30 sec.

72 °C – 30 sec.

Hold 4 °C – Indefinitely 4 °C – Indefinitely

Table 3. Oligonucleotide sequences used for quantitative polymerase chain reaction assays. 

[Oligo, oligonucleotide; NZMS, New Zealand mudsnail] 

Oligo ID Sequence Assay(s) used

NZMS-CYTB-F TGTTTCAAGTGTGCTGGTTTAYA NZMS-cytb, NZMS-cytb-mod, 

NZMS-cytb-coi

NZMS-CYTB-R CAAATGGRGCTAGTTGATTCTTT NZMS-cytb, NZMS-cytb-mod, 

NZMS-cytb-coi

NZMS-CYTB-PROBE 6FAMCCTCGACCAATATGTAAAT/MGB NZMS-cytb

NZMS-CYTB-EXT 56-FAM/ATYCCTCGA/ZEN/CCAATATGTAAATAAATAC/3IABkFQ NZMS-cytb-mod, 

NZMS-cytb-coi

NZMS-COI-F CTCCTGCTTTCTTTACCTGTCT NZMS-coi, NZMS-cytb-coi

NZMS-COI-R ACCAGAATAAGTGCTGATAAAGGAT NZMS-coi, NZMS-cytb-coi

NZMS-COI-FAM 56-FAM/ATTTCGATC/ZEN/CGTTAATAGTATTGTAATAGCYCC/3IABkFQ NZMS-coi

NZMS-COI-JOE 56-JOE/ATTTCGATC/ZEN/CGTTAATAGTATTGTAATAGCYCC/3IABkFQ NZMS-cytb-coi

NZMS-gBlock TATGGCTGGCTACTTCGTGCCCTACACGCAAATGGGGCTAGTTGATTC

TTTATTTGTATTTATTTACATATTGGTCGAGGAATATATTATGGATCAT

ATGTAAACCAGCACACTTGAAACATTGGAGTAATTCTTCTCGTTTTAA

CTATGGCCACTGCTTTTTTAGGTTATGTTTCTGTTAAAATTACAGCTAT

TTTACTCCTGCTTTCTTTACCTGTCTTAGCAGGGGCTATTACAATACTA

TTAACGGATCGAAATTTCAATACTGCTTTTTTTGATCCTGCTGGGGGTG

GTGATCCTATCCTTTATCAGCACTTATTCTGGTTTTTCGGACACCCAGA

AGTATATAT

All Asssays
5

DNA Sequencing

The products resulting from at least one positive reaction from an expected-positive 

sample and any positive reactions from expected-negative samples were sequenced for each 

assay at MCGL. The qPCR products were purified using the MultiScreen PCR μ96 filter plate 

(Millipore Corporation, Billerica, Mass.) according to the manufacturer’s protocol. The purified 

PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit 

(Applied Biosystems, Inc., Foster City, Calif.). Each cycle sequencing reaction mixture 

contained 0.75x proprietary concentration BigDye buffer (MCLAB, San Francisco, Calif.), 0.75 

μL BDX64 (BigDye enhancing buffer, MCLAB), 0.32 μM forward primer (NZMS-COI-F or 

NZMS-CYTB-F [table 3]), 0.25 μL BigDye v3.1 (Applied Biosystems Inc.), and 1.5 μL of 

cleaned PCR product in a final volume of 10 μL. Cycle sequencing reactions were purified with 

the Montage SEQ96 Sequencing Reaction Cleanup Kit (Millipore), and sequenced on an Applied 

Biosystems 3730xl DNA Analyzer (Applied Biosystems, Inc.) at the Molecular Conservation 

Genetics Laboratory. The sequences were manually edited in Geneious version 8.0.5 

(Drummond and others, 2012) and the species identities were determined with the National 

Center for Biotechnology Information (NCBI) nucleotide database and Megablast search 

algorithm (Altschul and others, 1997, as implemented in Geneious version 8.0.5). The highest 

percent sequence similarity obtained from the Basic Local Alignment Search Tool (BLAST) 

search (minimum percent match = 95.0 percent) was used to identify the likely source species for 

each qPCR sample sequenced.

Data Analysis

Baseline fluorescence was automatically normalized for all qPCR replicates within each 

plate by the software used at each laboratory. Threshold values were manually set to the same 

level across all plates for each assay targeting the start of the exponential phase of the

amplification plots. Positive detection was determined if the normalized fluorescence rose above 

the threshold values. Samples were considered positive for New Zealand mudsnail DNA with the 

single marker assays (NZMS-cytb, NZMS-cytb-mod, NZMS-coi) if any of the eight replicate 

reactions had positive detection. Samples were considered positive for New Zealand mudsnail 

DNA in the duplex assay (NZMS-cytb-coi) if both dyes had positive detection independently in 

any of the eight replicate reactions. Only the duplex assay was used to analyze the monitoring 

samples. Samples that had positive detection but failed sequence confirmation were considered 

negative for New Zealand mudsnail DNA.

The number of detections in known-positive, expected-positive, and expected-negative 

samples were compared across the four assays. Assays were considered validated if detection 

probability was equal to or higher than that of the published NZMS-cytb assay for positive 

samples with equal or less detections among negative samples.

Results

In total, 20 samples positive for New Zealand mudsnail DNA and 10 samples negative 

for New Zealand mudsnail DNA were analyzed using four qPCR assays at each of the two labs. 

All four assays at both labs detected New Zealand mudsnail DNA in all 20 positive samples 

(tables 4, 5). All four assays at both labs did not detect or were considered negative for New 

Zealand mudsnail DNA in all 10 expected-negative samples after sequencing confirmation 

(tables 4, 5). The sequences obtained are shown in table 6. All assays were considered validated, 
6

and the duplex assay (NZMS-cytb-coi) was selected as the preferred analysis for NZMS

monitoring because of the increased efficiency associated with analyzing two markers 

simultaneously. 

Table 4. Upper Midwest Environmental Sciences Center assay validation results.

(Please see accompanying file NZMS-Table4.xlsx) 

Table 5. Molecular Conservation Genetics Laboratory assay validation results.

(Please see accompanying file NZMS-Table5.xlsx) 

Table 6. Sequencing results.

(Please see accompanying file NZMS-Table6.xlsx) 

Triplicate water samples collected from 45 sites were analyzed at UMESC in eight 

replicate reactions using the NZMS-cytb-coi assay with additional replicate water samples 

collected from four sites being verified at the MCGL (table 1). All samples were negative for 

New Zealand mudsnail DNA. The results were verified in the four samples at the MCGL (fig. 1, 

table 7). All positive controls detected New Zealand mudsnail DNA and all negative controls did 

not detect New Zealand mudsnail DNA.
7

Figure 1. Map showing the locations of sampling sites. White circles, sites that tested negative for New 

Zealand mudsnail DNA; red circles, original infestation sites. Sites not shown because global positioning 

system coordinates were not recorded: North Bear Creek, Iowa; South Bear Creek, Iowa; Dell Creek, Wis.; 

Gordon Creek at Spring Creek Rd., Wis.; Gordon Creek at State Route 39, Wis.; Mt. Vernon Creek, Wis.; 

Rowan Creek, Wis.; Spring Creek, Wis.; Story Creek, Wis.; Sugar River at Frenchtown, Wis.; West Branch 

Sugar River, Wis.
8

Table 7. Monitoring results.

(Please see accompanying file NZMS-Table7.xlsx) 

References

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J., 

1997, Gapped BLAST and PSI-BLAST—A new generation of protein database search 

programs: Nucleic Acids Research, v. 25, p. 3389–3402. 

Drummond, A.J., Ashton, B., Buxton, S., Cheung, M., Cooper, A., Duran, C., Field, M., Heled, 

J., Kearse, M., Markowitz, S., Moir, R., Stones-Havas, S., Sturrock, S., Thierer, T., and 

Wilson, A., 2012, Geneious ver. 8.0.5: Biomatters, Ltd., available 

from http://www.geneious.com/. 

 Goldberg, C.S., Sepulveda, A., Ray, A., Baumgardt, J., Waits, L.P., 2013, Environmental DNA 

as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum): 

Freshwater Science, v. 32, no. 3, p. 792–800. 
9

Appendix Sample collection data sheet.
Merkes and others—Validation of eDNA Markers for New Zealand Mudsnail Surveillance and Initial eDNA Monitoring at Mississippi River Basin Sites—Open-File Report 2015-1037

ISSN 2331-1258 (online)

http://dx.doi.org/10.3133/20151037