Conventional Detection of Microalgaes Using Nuclease Protection Assay Sandwich Hybridization

Abstract

The frequent occurrence of harmful algal blooms (HABs) at worldwide has serious damages on the marine environment and fishery resources. A variety of harmful algae including Heterocapsa triquetra, Heterosigma akashiwo, Chattonella marina, Scrippsiella trochoidea have been revealed as causative sources of HABs in Korea’s coasts, but their existence have not been tested in large number of samples and wide scale of marine environment using conventional approaches including light and electron microscopy in long-term monitoring and high-throughput sampling projects. In this study, an integrated sandwich hybridization and nuclease protection assay (NPA-SH) was established for the quantitative detection of H. triqeutra, H. akashiwo, C. marina, S. trochoidea. The specificity of the probes was verified with individual and mixed cultures as well as field collection from Goseong Bay in South Korea. And the quantity of four microalgaes determined by NPA-SH analysis showed such a good correlation with cell-counting by a light microscope. In addition a standard curves for four microalgaes was demonstrated to represent the correlation between optical absorbance in the NPA-SH assay and cell density. These results show that the NPA-SH method is reliable, specific, and precise in the detection of H. triqeutra, H. akashiwo, C. marina, S. trochoidea. Thus, this approach provides an effective alternative to traditional, morphology-basecrippsiella trochoidea have been revealed as causative sources of HABs in Korea’s coasts, but their existence have not been tested in large number of samples and wide scale of marine environment using conventional approaches including light and electron microscopy in long-term monitoring and high-throughput sampling projects. In this study, an integrated sandwich hybridization and nuclease protection assay (NPA-SH) was established for the quantitative detection of H. triqeutra, H. akashiwo, C. marina, S. trochoidea. The specificity of the probes was verified with individual and mixed cultures as well as field collection from Goseong Bay in South Korea. And the quantity of four microalgaes determined by NPA-SH analysis showed such a good correlation with cell-counting by a light microscope. In addition a standard curves for four microalgaes was demonstrated to represent the correlation between optical absorbance in the NPA-SH assay and cell density. These results show that the NPA-SH method is reliable, specific, and precise in the detection of H. triqeutra, H. akashiwo, C. marina, S. trochoidea. Thus, this approach provides an effective alternative to traditional, morphology-base methods for the rapid, precise identification and quantification of harmful algal species and could be used to monitor phytoplankton in field surveys.