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

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.