Field-adaptable RAA-CRISPR/LbCas12a assay for specific detection of Karenia brevis in natural seawater

Karenia species occur widely and can trigger harmful algal blooms (HABs) that threaten coastal ecosystems, aquaculture, and human health. Karenia brevis (K. brevis), a brevetoxin-producing species, is of particular concern. Although large blooms have not been reported in mainland China, K. brevis may occur at low abundance and can co-occur with morphologically similar Karenia spp., necessitating species-level detection. Here, we established a rapid assay for K. brevis by integrating recombinase-aided amplification (RAA) with clustered regularly interspaced short palindromic repeats (CRISPR)-Lachnospiraceae bacterium Cas12a (LbCas12a). RAA primers and crRNAs were designed from the highly variable internal transcribed spacer (ITS) region to discriminate K. brevis from closely related Karenia species. Readouts included fluorescence using a quenched reporter (FQ) and lateral-flow detection using a FAM-biotin reporter (FB) with a lateral flow dipstick (LFD). A simple fast-lysis DNA preparation was compatible with the RAA, supporting field-adaptable workflows followed by either portable fluorescence viewing or FB-LFD readout. The FQ format also supports kinetic monitoring on laboratory instruments. The assay detected 5.9 × 10³ copies/μL of ITS plasmid and as low as 1 cell/mL in seawater (0.1 cell-equivalents per reaction input) with no cross-reaction to nine non-target algal species. Natural HAB water collected in Lianjiang, Fujian (May 2024) was tested negative for K. brevis by the assay and by cloning/phylogenetic analysis, but became positive after spiking with K. brevis cells, indicating minimal seawater inhibition. This platform provides sensitive and specific detection of rapid K. brevis and is readily extendable to other closely related Karenia species.