Multiplex assay to identify eggs of three fish species from the northern Gulf of Mexico, using locked nucleic acid Taqman real-time PCR probes

Abstract

Understanding the early life histories of economically and ecologically important fish species is vital to developing effective management strategies. While adult-derived egg production estimates are important to fisheries management models, determining fish egg abundance in the wild is cumbersome and oftentimes impossible, as most fish eggs cannot be morphologically identi-fied to species. Here, we report on the development of a real-time polymerase chain reaction assay to identify the eggs of 3 important Gulf of Mexico fish species (red drum Sciaenops ocellatus, red snapper Lutjanus campechanus and vermilion snapper Rhomboplites aurorubens). This single-tube, single-egg assay uses 3 species-specific fluorescently labeled locked nucleic acid (LNA) Taqman probes to identify DNA extracted from individual eggs of the 3 targeted species. To ensure that the probes were truly species specific, the LNA Taqman assay was tested against DNA from 25 or more known adults of each target species as well as DNA from 62 non-target northern Gulf of Mexico fishes. All DNA extracts from the 3 target species showed the appropriate positive reaction from the assay, while all 62 other DNA extracts showed null reactions. The assay was also tested individually against 60 fertilized S. ocellatus eggs and the same number of unfertilized L. campechanus and R. aurorubens eggs, resulting in positive identification in all cases. By running this LNA Taqman assay on a large number of field-collected eggs, it is possible to quickly and positively estimate field egg abundances, adding a powerful tool for improving ichthyoplankton assessments and determining fine-scale temporal and spatial spawning distributions. © Inter-Research 2008.