• Journal of Life Science
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• v.30 no.1
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• pp.26-31
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• 2020

Heterosigma akashiwo is a globally distributed raphidophyte that forms blooms and causes significant losses to the aquaculture industry in many coastal countries. The development of a fast and sensitive detection method is therefore required to facilitate the appropriate warning of harmful algal blooms. In this study, a nuclease protection integrated with sandwich hybridization (NPA-SH) assay was developed to both qualitatively and quantitatively detect H. akashiwo. The NPA, capture and signal probes were designed by nucleotide sequencing of H. akashiwo. The applicability of NPA-SH was evaluated using cultured H. akashiwo cells and field samples collected at Goseong Bay, Korea. The results show that this method has good applicability and effectiveness in analyzing cultured cells and field samples. A linear regression equation for the quantitative analysis of H. akashiwo was obtained, and the lower detection limit of the assay was 1×10⁴ cells/ml. There was no statistically significant difference in the results of H. akashiwo quantitation using NPA-SH compared to those obtained using a microscope. These results indicate that NPA-SH can be a good alternative to the traditional microscopic method used to monitor H. akashiwo.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.8
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• pp.627-633
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• 2019

Rapid and accurate identification of marine microalgae causing harmful algal blooms (HABs) is a crucial 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 the 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. The treatment time and appropriate amounts of the probe and sucrose that optimized the reaction were determined. As a result, the developed INV-SH reduced the treatment time in the field compared with NPA-SH, and also enabled the use of 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.