Molecular techniques for the identification of freshwater fish species for environmental monitoring programs

Abstract

Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time PCR (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 probe for the identification of eight freshwater fish species. The development of a fully automated species-decoder algorithm allowed for target species identification with 100% accuracy while completely removing any false-positive detection of non-target species. Furthermore, the probe-based qPCR technique utilized in this study is substantially more cost-effective and time efficient than DNA barcoding and morphological identification methods. The qPCR assays were also highly sensitive and accurately detected target species from collected environmental DNA (eDNA) samples. 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.