Rapid Identification of Adult and Naupliar Stages of Copepods Using DNA Hybridization Methodology

Abstract:

Larval stages of common marine invertebrates and their ecological roles within their respective communities are frequently ignored because they are hard to identify. Morphological characters are often insufficient to differentiate between genera, much less species. To overcome the obstacles associated with species identification of copepod larvae, we developed a microtiter plate-based hybridization assay. Species-specific probes based on rDNA sequences were bound to microplates and used to capture target DNA. A novel method of linking the probes to the plate with poly-T tail ensured the probes were positioned above the plate surface and available for hybridization; this significantly increased the sensitivity of the assay. Target DNA extracted from individual copepods was amplified with biotin-labeled primers. The labeled target DNA bound to the probe specific for that species and produced a colorimetric change in the assay. The assay can be rapidly performed on freshly caught or ethanol preserved samples and the results visually interpreted.