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http://dx.doi.org/10.5352/JLS.2020.30.1.26

Development of a Nuclease Protection Assay With Sandwich Hybridization (NPA-SH) to Monitor Heterosigma akashiwo  

Kang, Mingyeong (Ecological Risk Research Division, Korea Institute of Ocean Science and Technology)
Park, Mirye (Bioresources Culture Collection Division, Nakdonggang National Institute of Biological Resources)
Kim, Kang Eun (Ecological Risk Research Division, Korea Institute of Ocean Science and Technology)
Lee, Taek-Kyun (Ecological Risk Research Division, Korea Institute of Ocean Science and Technology)
Publication Information
Journal of Life Science / v.30, no.1, 2020 , pp. 26-31 More about this Journal
Abstract
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.
Keywords
Algal blooms; field monitoring; HABs; Heterosigma akashiwo; NPA-SH;
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1 Tyrrell, J. V., Bergquist, P. R., Bergquist, P. L. and Scholin, C. A. 2001. Detection and enumeration of Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae) using rRNA-targeted oligonucleotide probes. Phycologia 40, 457-467.   DOI
2 Tyrrell, J. V., Connell, L. B. and Scholin, C. A. 2002. Monitoring for Heterosigma akashiwo using a sandwich hybridization assay. Harmful Algae 1, 205-214.   DOI
3 Venugopalan, C. and Kapoor, H. C. 1997. Single step isolation of plant RNA. Phytochemistry 46, 1303-1305.   DOI
4 Woelfl, S. and Whitton, B. A. 2000. Sampling, preservation and quantification of biological samples from highly acidic environments ($pH{\leq}3$). Hydrobiologia 433, 173-180.   DOI
5 Yu, Z., Tiezhu, M. and Zhigang, Y. 2008. Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay (NPA-SH). J. Environ. Sci. 20, 1481-1486.   DOI
6 Zhang, C., Wang, Y., Guo, C., Chen, G., Kan, G., Cai, P. and Zhou, J. 2018. Comparison of loop-mediated isothermal amplification with hyperbranched rolling circle amplification as a simple detection method for Heterosigma akashiwo. Harmful Algae 73, 1-11.   DOI
7 Zhen, Y., Mi, T. and Yu, Z. 2009. Detection of several harmful algal species by sandwich hybridization integrated with a nuclease protection assay. Harmful Algae 8, 651-657.   DOI
8 Zhen, Y., Yu, Z., Cai, Q., Mi, T. and Li, R. 2007. Detection of two diatoms using sandwich hybridization integrated with nuclease protection assay (NPA-SH). Hydrobiologia 575, 1-11.   DOI
9 Blanco, E. P., Hagstrom, J., Salomon, P. S. and Graneli, E. 2013. Detection of Heterosigma akashiwo (Hada) using specific RNA probes: variability of RNA content with environmental conditions. Harmful Algae 24, 80-88.   DOI
10 Bowers, H. A., Tomas, C., Tengs, T., Kempton, J. W., Lewitus, A. J. and Oldach, D. W. 2006. Raphidophyte [Chadefaud ex Silva] systematics and rapid identification: sequence analyses and real-time PCR assays. J. Phycol. 42, 1333-1348.   DOI
11 Main, C. R., Greenfield, D. I., Doll, C., Wang, Y., Whereat, E. B., Mortensen, R., Pettay, D. T. and Coyne, K. J. 2018. Critical comparison of molecular methods for detection and enumeration of the harmful algal species, Heterosigma akashiwo, in environmental water samples. J. Appl. Phycol. 30, 2425-2434.   DOI
12 Cai, Q., Li, R., Zhen, Y., Mi, T. and Yu, Z. 2006. Detection of two Prorocentrum species using sandwich hybridization integrated with nuclease protection assay. Harmful Algae 5, 300-309.   DOI
13 Chen, G. F., Wang, G. C., Zhang, C. Y., Zhang, B. Y., Wang, X. K. and Zhou, B. C. 2008. Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae). J. Exp. Mar. Biol. Ecol. 355, 66-75.   DOI
14 Doll, C., Main, C. R., Bianco, C., Coyne, K. J. and Greenfield, D. I. 2014. Comparison of sandwich hybridization assay and quantitative PCR for the quantification of live and preserved cultures of Heterosigma akashiwo (Raphidophyceae). Limnol. Oceanogr. Meth. 12, 230-243.
15 Ebenezer, V., Medlin, L. K. and Ki, J. S. 2012. Molecular detection, quantification, and diversity evaluation of microalgae. Mar. Biotechnol. 14, 129-142.   DOI
16 Keppler, C. J., Hoguet, J., Smith, K., Ringwood, A. H. and Lewitus, A. J. 2005. Sublethal effects of the toxic alga Heterosigma akashiwo on the southeastern oyster (Crassostrea virginica). Harmful Algae 4, 275-285.   DOI
17 Park, M., Park, S. Y., Hwang, J., Jung, S. W., Lee, J., Chang, M. and Lee, T. K. 2018. Integration of the nuclease protection assay with sandwich hybridization (NPA-SH) for sensitive detection of Heterocapsa triquetra. Acta Oceano. Sin. 37, 107-112.
18 Suh, S. S., Park, M., Hwang, J., Kil, E. J., Lee, S. and Lee, T. K. 2015. Detection of the dinoflagellate, Cochlodinium polykrikoides, that forms algal blooms using sandwich hybridization integrated with nuclease protection assay. Biotechnol. Lett. 38, 57-63.