Detection of Cochlodinium polykrikoides using sandwich hybridization integrated with nuclease protection assay

Abstract

The frequent occurrence of harmful algal blooms (HABs) worldwide has serious impacts on the marine environment and fishery resources. Cochlodinium polykrikoides is an important causative agent of HABs in Korea’s seas, but the presence of this organism cannot be tested in large number of samples using conventional approaches including light and electron microscopy in long-term monitoring and high-throughput sampling projects. As such, in this study, an integrated sandwich hybridization and nuclease protection assay (NPA-SH) was established for the quantitative detection of C. polykrikoides. The specificity of the probes was verified with individual and mixed cultures as well as field collection from Gosung Bay, Korea, and the quantity of C. polykrikoides determined by NPA-SH analysis showed a good correlation with that determined by cell-counting with a light microscope. In addition a standard curve for C. polykrikoides was established to represent the correlation between optical absorbance in the NPA-SH assay and cell density. The results show that the NPA-SH method is reliable, specific, and accurate in the detection of C. polykrikoides thus, this approach provides an efficient alternative to traditional, morphology-based methods for the rapid identification and quantification of harmful algal species and could be used to monitor phytoplankton in field surveys.his organism cannot be tested in large number of samples using conventional approaches including light and electron microscopy in long-term monitoring and high-throughput sampling projects. As such, in this study, an integrated sandwich hybridization and nuclease protection assay (NPA-SH) was established for the quantitative detection of C. polykrikoides. The specificity of the probes was verified with individual and mixed cultures as well as field collection from Gosung Bay, Korea, and the quantity of C. polykrikoides determined by NPA-SH analysis showed a good correlation with that determined by cell-counting with a light microscope. In addition a standard curve for C. polykrikoides was established to represent the correlation between optical absorbance in the NPA-SH assay and cell density. The results show that the NPA-SH method is reliable, specific, and accurate in the detection of C. polykrikoides thus, this approach provides an efficient alternative to traditional, morphology-based methods for the rapid identification and quantification of harmful algal species and could be used to monitor phytoplankton in field surveys.