Rapid Detection of Cochlodinium polykrikoides by Sandwich Hybridization Integrated with Invertase Assay

Abstract

Rapid and accurate identification of marine microalgae causing harmful algal blooms (HABs) is a very important tool for predicting and managing HABs. We previously developed a nuclease protection assay sandwich hybridization (NPA-SH) method for the in situ detection of blooming microalgae Cochlodinium polykrikoides. In this study, we improved the applicability of the NPA-SH method for the detection of C. polykrikoides by simplifying the reaction step. For this purpose, invertase (INV) was conjugated to signal probe instead of using fluorescence, and sucrose was used as a reactant to induce a color reaction. The INV-signal probe conjugation was confirmed by SDS-PAGE and epifluoromicroscopy, and the amounts of the appropriate probe and sucrose, and the treatment time for optimizing the reaction were determined. As a result, the developed INV-SH reduced the treatment time in the field compared with NPA-SH, and it was possible to use a relatively small volume and low-priced personal glucose meter as well as an absorbance meter. INV-SH is the first C. polykrikoides species identification technology to which INV has been applied and could be an improved field technique.