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Development of a Rapid Detection Method to Prorocentrum lima by Loop-Mediated Isothermal Amplification with Hydroxy Naphthol Blue

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Abstract

Prorocentrum lima, a widely distributed dinoflagellate known for its production of harmful biotoxins, poses a significant threat to humans, aquaculture, and marine ecosystems. As a result, the detection of this toxic alga in coastal waters has become an urgent research focus. In this study, a rapid, sensitive, and cost-effective detection method based on loop-mediated isothermal amplification (LAMP) was developed to identify P. lima. In this method, cell extracts of P. lima were diluted and used directly as templates for amplification, eliminating the need for nucleic acid purification and simplifying the detection process. Hydroxy naphthol blue (HNB) was incorporated into the reaction mix to facilitate result interpretation, enabling visual determination of the amplification outcome with the naked eye. The entire detection process, from DNA extraction to template amplification and product detection, could be completed within 80 min using a simple constant temperature-control device. This LAMP-based detection method demonstrated excellent reliability, specificity, and a low detection limit of 5.87 cells/mL for DNA crude extract. The assay offered an efficient alternative to PCR for rapid detection of P. lima. By streamlining the detection process and offering a visual readout, this technique holds promise for efficient and routine monitoring of harmful algal species, benefitting both research efforts and environmental management strategies.

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Acknowledgements

We would like to thank Professor Aifeng Li from the Ocean University of China for providing algae used in the experiment.

Funding

This work was supported by the National Key Research and Development Program of China (No. 2022YFC3105202), the Scientific and Technological Innovation Project of the Laoshan Laboratory (No. LSKJ202203700), and the National Nature Science Foundation of China (No. 41976133).

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Authors and Affiliations

  1. Key Laboratory of Marine Environment and Ecology, College of Environmental Science and Engineering, Ocean University of China, Ministry of Education, Qingdao, 266100, China

    Chao Yang, Yu Zhen & Tiezhu Mi

  2. Laboratory for Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, 266237, China

    Yu Zhen & Tiezhu Mi

  3. Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China, Qingdao, 266100, China

    Yu Zhen & Tiezhu Mi

  4. Weifang Bangmao Biotechnology Co., Ltd., Weifang, 261000, China

    Jialin Hou

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  1. Chao Yang
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  2. Yu Zhen
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  4. Tiezhu Mi
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Contributions

Chao Yang, Yu Zhen, and Jialin Hou contributed to conceptualization of this study. Yu Zhen obtained the fundings. Investigation, methodology, data curation, formal analysis, and writing-original manuscript were performed by Chao Yang. Yu Zhen, Jialin Hou, and Tiezhu Mi contributed to the writing-review and supervision. Yu Zhen and Jialin Hou contributed to the editing of manuscript. All authors contributed to the article and approved the submitted version.

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Correspondence to Yu Zhen.

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Yang, C., Zhen, Y., Hou, J. et al. Development of a Rapid Detection Method to Prorocentrum lima by Loop-Mediated Isothermal Amplification with Hydroxy Naphthol Blue. Mar Biotechnol (2024). https://doi.org/10.1007/s10126-024-10310-2

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