Determining factors that influence the molecular quantification of the harmful raphidophyte Heterosigma akashiwo using a sandwich hybridization assay

Abstract

Molecular approaches for detecting and quantifying harmful algal bloom (HAB) species have become more commonplace because of their capability to distinguish taxa and species. Sandwich hybridization assay (SHA) is a molecular probe technique that uses two DNA probes to detect species or taxon-specific large subunit rRNA sequences and can be used to quantify cell abundances in environmental samples without nucleic acid purification or amplification. However, the influence of certain physiological and methodological factors on SHA optical density (OD) remains unclear. Elucidating such factors will help in evaluating SHA's reliability as a HAB management tool. The following specific factors were addressed; (1) do HAB strains from geographically distinct populations react differently on the SHA, (2) are samples preserved in Lugol's iodine solution stable enough for SHA quantification, (3) does algal growth phase or diel cycle influence SHA OD, and (4) how do nutrient or light limitation affect SHA OD? All experiments were carried out in the laboratory using the globally-distributed, ichthyotoxic raphidophyte Heterosigma akashiwo as the study species because SHA has been developed and rigorously validated for it. Results showed that SHA standard curves for some of the strains did not vary significantly from previously published results, but a few strains did display distinct reactions. Samples preserved in Lugol's were quantifiable for a week at room temperature and up to four months when refrigerated. SHA OD declined as a culture progressed from exponential to stationary and decline phases, and displayed a diel trend. Nitrogen limitation had a significant influence on SHA OD whereas low phosphorus and light did not.