Open AccessDevelopment and validation of probe-based multiplex real-time PCR assays for the rapid and accurate detection of freshwater fish species
Author(s) -
Emily N. Hulley,
Sujeenthar Tharmalingam,
Andrew M. Zarnke,
Douglas R. Boreham
Publication year - 2019
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0210165
Subject(s) - biology , coregonus clupeaformis , primer (cosmetics) , micropterus , multiplex , bass (fish) , zoology , fishery , genetics , chemistry , fish <actinopterygii> , organic chemistry
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.