Abstract
Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time quantitative polymerase chain reaction (qPCR) provides an accurate, cost-effective and high-throughput method for species identification. Here we present the development and validation of species-specific primers and probes for the cytochrome c oxidase (COI) gene for the identification of eight ecologically and economically important freshwater fish species: lake whitefish (Coregonus clupeaformis), yellow perch (Perca flavescens), rainbow smelt (Osmerus mordax), brook trout (Salvelinus fontinalis), smallmouth bass (Micropterus dolomieu), round whitefish (Prosopium cylindraceum), spottail shiner (Notropis hudsonius) and deepwater sculpin (Myoxocephalus thompsonii). In order to identify novel primer-probe sets with maximum species-specificity, two separate primer-probe design criteria were employed. Highest ranked primer-probe sets from both methods were assayed to identify sequences that demonstrated highest specificity. Specificity was determined using control species from same genus and non-target species from different genus. Selected primer-probe sets were optimized for annealing temperature and primer-probe concentrations to identify minimum reagent parameters. The selected primer-probe sets were highly sensitive, with DNA concentrations as low as 1 ng adequate for positive species identification. A decoder algorithm was developed based on the cumulative qPCR results that allowed for full automation of species identification. Blinded experiments revealed that the combination of the species-specific primer/probes sets with the automated species decoder resulted in target species identification with 100% accuracy. We also conducted a cost/time comparison analysis between the qPCR assays established in this study with other species identification methods. The qPCR technique was the most cost-effective and least time consuming method of species identification. In summary, probe-based multiplex qPCR assays provide a rapid and accurate method for freshwater fish species identification, and the methodology established in this study can be utilized for various other species identification initiatives.
Highlights
Rapid and accurate species identification techniques are important for environmental monitoring programs, in industrial settings
This was achieved by obtaining c oxidase (COI) gene sequence information for the target species and their respective control species from the same genus (CON) using Barcode of Life Database (BOLD) and FishBase
The combination of the species-specific primer/probes sets with an automated species decoder algorithm resulted in target species identification with 100% accuracy coupled with complete absence of false-positive detection from non-target controls
Summary
Rapid and accurate species identification techniques are important for environmental monitoring programs, in industrial settings Large scale operations such as power plants, paper mills, petrochemical plants, petroleum refineries, natural gas and food processing plants negatively impact surrounding bodies of water due to use of once-through cooling systems. Morphological based species identification technique has often been the traditional method used by environmental monitoring programs to identify impinged fish species at intake points in once-through cooling operations. This technique has been proven to have several limitations, with impinged specimens that are often damaged or missing key identification traits. Ecological and environmental studies require a high level of specificity and accuracy, requiring individuals to be identified to genus- and species-level [5]
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