DOI QR코드

DOI QR Code

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

Invertase 분석이 통합된 sandwich hybridization에 의한 Cochlodinium polykrikoides의 신속 검출

  • Kang, Mingyeong (Ecological Risk Research Division, Korea Institute of Ocean Science and Technology) ;
  • Park, Mirye (Microbial Research Department, Nakdonggang National IOnstitute of Biological Resources) ;
  • Lee, Taek-Kyun (Ecological Risk Research Division, Korea Institute of Ocean Science and Technology)
  • 강민경 (한국해양과학기술원 생태위해성연구부) ;
  • 박미례 (국립낙동강생물자원관 미생물실) ;
  • 이택견 (한국해양과학기술원 생태위해성연구부)
  • Received : 2019.05.08
  • Accepted : 2019.08.02
  • Published : 2019.08.31

Abstract

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.

유해조류 대발생(harmful algal blooms, HABs)을 유발하는 해양 미세조류의 신속하고 정확한 종판별은 HABs을 예측하고 관리하기 위한 매우 중요한 도구이다. 우리는 이전 연구에서 적조 유발 미세조류인 Cochlodinium polykrikoides를 현장에서 검출하기 위한 nuclease protection assay sandwich hybridization (NPA-SH) 방법을 개발한 바 있다. 본 연구에서는 C. polykrikoides 검출을 위한 NPA-SH 방법의 반응 단계를 간소화시켜 현장 적용성을 향상시키는 것을 목적으로, signal probe에 형광 대신 invertase (INV)를 결합시켰으며, hybridization 과정에서 sucrose를 반응물로 사용하여 발색 반응을 유도하였다. INV와 signal probe의 결합 여부는 SDS-PAGE와 형광 현미경을 사용하여 확인하였으며, 반응 최적화를 위한 적정 프로브의 양, sucrose의 양 및 처리시간 등을 정립하였다. 본 연구의 결과 개발된 INV-SH는 NPA-SH와 비교하여 현장에서의 처리시간이 감소되었으며, 흡광도 측정기 뿐만 아니라, 상대적으로 부피가 작고 값 싼 개인 혈당계 사용이 가능하게 되었다. 본 연구에서 개발된 INV-SH는 INV가 적용된 최초의 C. polykrikoides 종판별 기술이며, 증진된 현장기술이 될 수 있다.

Keywords

References

  1. J. V. Tyrrell, L. B. Connell and C. A. Scholin. "Monitoring for Heterosigma akashiwo using a sandwich hybridization assay" Harmful Algae, vol. 1, no. 2, pp. 205-214, 2002. DOI: https://doi.org/10.1016/S1568-9883(02)00012-4
  2. K. Ayers, L. L. Rhodes, J. Tyrrell, M. Gladstone and C. Scholin. "International accreditation of sandwich hybridisation assay format DNA probes for microalgae" New Zealand J. Marine and Freshwater Research, vol. 39, no. 6, pp. 1225-1231, 2005. DOI: https://doi.org/10.1080/00288330.2005.9517388
  3. S. Diercks, L. K. Medlin and K. Metfies. "Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay" Harmful Algae, vol. 7, no. 2, pp. 137-145, 2008. DOI: https://doi.org/10.1016/j.hal.2007.06.005
  4. Q. Cai, R. Li, Y. Zhen, T. Mi and Z. Yu. "Detection of two Prorocentrum species using sandwich hybridization integrated with nuclease protection assay" Harmful Algae, vol. 5, no. 3, pp. 300-309, 2006. DOI: https://doi.org/10.1016/j.hal.2005.08.002
  5. Y. Zhen, T. Mi and Z. Yu. "Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay (NPA-SH)" J. Environmental Science, vol. 20, no. 12, pp. 1481-1486, 2008. DOI: https://doi.org/10.1016/S1001-0742(08)62553-X
  6. Y. Zhen, T. Mi and Z. Yu. "Detection of several harmful algal species by sandwich hybridization integrated with a nuclease protection assay" Harmful Algae, vol. 8, np. 5, pp. 651-657, 2009. DOI: https://doi.org/10.1016/j.hal.2008.12.001
  7. S. S. Suh, M. Park, J. Hwang, E. J. Kil, S. Lee and T.-K. Lee. "Detection of the dinoflagellate, Cochlodinium polykrikoides, that forms algal blooms using sandwich hybridization integrated with nuclease protection assay" Biotechnology Letters, vol. 38, no. 1, pp. 57-63, 2016. DOI: https://doi.org/10.1007/s10529-015-1947-5
  8. M. Park, S. Y. Park, J. Hwang, S. W. Jung, J. Lee, M. Chang and T.-K. Lee. "Integration of the nuclease protection assay with sandwich hybridization (NPA-SH) for sensitive detection of Heterocapsa triquetra " Acta Oceanologica Sinica, vol. 37, no. 5, pp. 107-112, 2018. DOI: https://doi.org/10.1007/s13131-018-1167-7
  9. Y. Xiang and Y. Lu. "Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets" Nature Chemistry, vol. 3, pp. 697-703, 2011. doi: https://doi.org/10.1038/nchem.1092.
  10. J. Su, J. Xu, Y. Chen, Y. Xiang, R. Yuan and Y. Chai. "Personal glucose sensor for point-of-care early cancer diagnosis" Chemical Communications, vol. 48, pp. 6909-6911, 2012. DOI: https://doi.org/10.1039/c2cc32729e
  11. Y. Xiang and Y. Lu. "An invasive DNA approach toward a general method for portable quantification of metal ions using a personal glucose meter" Chemical Communications, vol. 49, pp. 585-587, 2013. DOI: https://doi.org/10.1039/c2cc37156a
  12. X. Zhu, H. Xu, R. Lin, G. Yang, Z. Lin and G. Chen. "Sensitive and portable detection of telomerase activity in HeLa cells using the personal glucose meter" Chemical Communications, vol. 50, pp. 7897-7899, 2014. DOI: https://doi.org/10.1039/c4cc03553d
  13. K. S. Park, C. Y. Lee and H. G. Park. "Target DNA induced switches of DNA polymerase activity" Chemical Communications, vol. 51, pp. 9942-9945, 2015. DOI: https://doi.org/10.1039/C5CC02060C
  14. J. Bai, L. Liu, Y. Han, C. Jia and C. Liang. "One-step detection of hexokinase activity using a personal glucose meter" Analytical Methods, vol. 10, pp. 2075-2080, 2018. DOI: https://doi.org/10.1039/c8ay00498f
  15. H. Huang, G. Zhao and W. Dou. "Portable and quantitative point-of-care monitoring of Escherichia coli O157:H7 using a personal glucose meter based on immunochromatographic assay" Biosensors Bioelectronics, vol. 107, pp. 266-271, 2018. DOI: https://doi.org/10.1016/j.bios.2018.02.027