Development of a rapid RPA-CRISPR/Cas12a-LFD assay for the detection of toxin-producing harmful algal bloom species
ID:35
Oral Presentation
2027-01-13 15:35 (China Standard Time)
Session:Session 2 - Harmful Algal Blooms in the Asia Pacific: Recent Advances in Taxonomy, Biodiversity, Ecophysiology, Rapid Detection, and Early Warning to Mitigate Impacts of Coastal Ecosystem Health and Seafood Safety
Abstract
Harmful algal blooms (HABs) pose severe threats to marine ecosystems, aquaculture productivity, and public health worldwide, yet rapid on-site detection for toxigenic species remains technically challenging. Here, we developed a diagnostic assay integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a and lateral flow dipstick (LFD) for the visual detection of Pseudo-nitzschia multistrata, a toxigenic diatom species responsible for the production of neurotoxin domoic acid (DA). The assay targets the dabA gene, a functionally critical component of the DA biosynthesis gene cluster, whose detection serves as a molecular proxy for the toxigenic potential of the species. Following a simple alkaline lysis step, the released DNA was subjected to RPA for pre-amplification of the dabA gene at 39℃ for 35 minutes. The RPA product then served as the substrate for CRISPR/Cas12a reactions, in which the Cas12a protein, guide RNA, and a fluorescently labeled ssDNA probe were incubated at 37℃ for 30 min. Cas12a specifically recognized the target amplicon and activated its trans-cleavage activity, resulting in the cleavage of the probe. The RPA-CRISPR products were subsequently applied to LFD strips, with results visually interpreted within 5 min. The entire workflow, from cell lysis to result readout, can be accomplished within 1.5 hours without requiring thermal cycling equipment. This method offers a rapid, equipment-independent, and user-friendly approach for the detection of toxigenic HAB species, providing a technical foundation for early warning and routine monitoring of HAB events in coastal ecosystems.
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Jan 12–15
2027
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