Computational protocol: The Relationship between the Distribution of Common Carp and Their Environmental DNA in a Small Lake

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Protocol publication

[…] Although two C. carpio qPCR assays had been developed prior to this study , , a screen against the NCBI database indicated potential non-specific amplification of non-target fish species (). Therefore, a qPCR assay was developed for the current study. Four genes were considered in the development of a novel qPCR marker specific to the common carp: (1) mitochondrial gene cytochrome b, (2) mitochondrial gene cytochrome c oxidase subunit 1, (3) mitochondrial gene control (D-loop) region, and (4) the nuclear gene recombination activating gene 1 (RAG1). Candidate primer sets were identified by NCBI Primer-BLAST using sequences under GenBank accession number X61010.1 for mtDNA and EF458304.1 for the RAG1 gene. Specificity was initially screened against the BLASTn database sequences for 15 fish species (). Minor groove binder (MGB) probes were manually designed using the Primer Probe Test Tool in Primer Express Software v3.0.1 (Life Technologies, Grand Island, NY). Assays with amplification efficiency outside the range of acceptable values of 90–110% or a limit of detection above 300 copies per reaction were not considered. We defined the limit of detection (LOD) as the lowest value at which three replicate reactions would successfully amplify with a quantification cycle (Cq) value of less than 40 cycles within the linear range of the standard curve.Candidate markers were screened for specificity for carp by testing for amplification of 15 ng of fin clip DNA from carp and 34 native and non-native fish species (). Fin clip samples for genetic marker specificity testing were extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and assayed as described below.Next, we tested markers using aqueous samples. Three 340 L flow-through tanks were set up to confirm the ability of the marker to detect carp eDNA. Prior to this experiment, all tanks were treated with 10% bleach for 30 minutes to remove all traces of DNA. The flow through rate was set at 600 mL/min, and temperature was maintained at 18°C. The first tank was stocked with 10 carp (35 g), and the second tank was stocked with 10 goldfish (Carassius auratus) (50 g), while the third tank was stocked with five fish of both species. These stocking levels corresponded to a biomass of 0, 438, and 875 mg/L of carp. Fish were fed once daily ad libitum a combination of flake feed (Color Tropical Marine Flake, Pentair Aquatic Eco-systems, Inc., Apopka, FL) and 2.5 mm pellet feed (Oncor Fry, Skretting USA, Tooele, UT) that did not contain target genetic markers. After 6 days, 4 1 L water samples were collected from each tank, immediately stored at 4°C, and filtered within 4 h. Molecular analyses followed protocols described below. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for care and holding of laboratory fish was approved by the University of Minnesota’s Institutional Animal Care and Use Committee (IACUC) (Protocol: 1407-31659A). No anesthesia or euthanasia was required as part of this study. […]

Pipeline specifications

Software tools Primer-BLAST, BLASTN, Primer Express
Application qPCR
Organisms Cyprinus carpio, Hemisus marmoratus