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Development of a recombinase polymerase amplification combined with lateral flow dipstick assay for rapid and sensitive detection of Heterosigma akashiwo

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Abstract

Heterosigma akashiwo is one of the main toxin-producing species that is globally known for frequently forming fish-killing harmful algal blooms. In this study, a novel technique referred to as recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) (RPA-LFD) was established for detection of H. akashiwo. RPA primers and an LFD probe were designed based on the sequence of the large ribosomal subunit (LSU rDNA) D1-D2 region of H. akashiwo. RAP/RPA-LFD was experimentally verified to be specific, displaying no cross-reaction with other control microalgae. It was demonstrated that the optimal amplification temperature and time for RPA were 41 °C and 40 min. The LFD assay was completed in only 5–10 min to analyze RPA products, and RPA results could be visualized directly. RPA-LFD was 100 times more sensitive than PCR, displaying a detection limit of 3.37 × 10−4 ng µL−1 for genomic DNA of H. akashiwo and 1.28 × 102 copies µL−1 for the recombinant plasmid containing the inserted LSU rDNA D1-D2 region of H. akashiwo. In this study, RPA-LFD’s detection limit was 1 cell mL−1, which is feasible to apply in the field test. Thus, the established RPA-LFD targeting the detection of H. akashiwo is characterized by simplicity, rapidity, sensitivity, specificity, and visualization.

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Funding

This work was financially supported by Shandong Provincial Natural Science Foundation, China (ZR2020MD081); the National Scientific Foundation of China (Nos. 31600309, 41476086); HIT Scientific Research Innovation Fund/the Fundamental Research Funds for the Central Universities (Nos. HIT.NSRIF.201702 and HIT.NSRIF.201709); and HIT Environment and Ecology Innovation Special Funds (No. HSCJ201622).

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  1. College of Oceanology, Harbin Institute of Technology (Weihai), Weihai, 264209, People’s Republic of China

    Bingkun Wang, Chunyun Zhang, FuQueryguo Liu, Runqi Li, Yuanyuan Wang & Guofu Chen

  2. School of Environment, Harbin Institute of Technology, Harbin, 264209, People’s Republic of China

    Bingkun Wang, Chunyun Zhang, FuQueryguo Liu & Guofu Chen

  3. School of Marine Science and Technology, Harbin Institute of Technology At Weihai, Wenhua West Road, Weihai, Shandong Province, 2#, China

    Chunyun Zhang & Guofu Chen

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Wang, B., Zhang, C., Liu, F. et al. Development of a recombinase polymerase amplification combined with lateral flow dipstick assay for rapid and sensitive detection of Heterosigma akashiwo. J Appl Phycol 33, 3165–3178 (2021). https://doi.org/10.1007/s10811-021-02538-5

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