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Open-File Report 2015–1037

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

Thumbnail of and link to report PDF (263 KB)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 expected-negative 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 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.

First posted March 3, 2015

  • Tables 4-7 XLSX (114 KB)
    Table 4. Upper Midwest Environmental Sciences Center assay validation results. (30 KB)

    Table 5. Molecular Conservation Genetics Laboratory assay validation results. (30 KB)

    Table 6. Sequencing results. (20 kB)

    Table 7. Monitoring results. (34 KB)

For additional information, contact:
Director, Upper Midwest Environmental Sciences Center
U.S. Geological Survey
2630 Fanta Reed Road
La Crosse, Wisconsin 54603
Phone: 608 783–6451
http://www.umesc.usgs.gov/

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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., https://dx.doi.org/10.3133/ofr20151037.

ISSN 2331-1258 (online)



Contents

Abstract

Introduction

Materials and Methods

Results

References

Appendix - Sample collection data sheet

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