Exponential rolling circle amplification coupled with lateral flow dipstick strips as a rapid and sensitive method for the field detection of Karlodinium veneficum

Harmful algal blooms (HABs) caused by microalgae pose a great threat to human health, marine ecosystems, tourism, and aquaculture worldwide. Efficient, sensitive, and simple methods for the detection of harmful microalgal species must be developed to reduce the economic losses and damages caused by HABs. This study successfully established a novel method for the detection of the globally distributed HAB-forming species Karlodinium veneficum. The developed method was based on exponential rolling circle amplification (E-RCA) coupled with lateral flow dipstick (LFD) strips. A special oligonucleotide sequence-padlock probe (PLP) was designed on basis of the molecular cloning and subsequent sequencing of the D1–D2 region of the large subunit ribosomal DNA (LSU rDNA) of K. veneficum. The E-RCA reaction system was then established. E-RCA products could be visually analyzed through 2% agarose gel electrophoresis and the LFD assay. The optimized E-RCA conditions were as follows: PLP concentration of 20 pM, ligation cycle number of 12, ligation temperature of 56 °C, amplification duration of 45 min, and amplification temperature of 61 °C. The developed E-RCA-LFD assay was specific for K. veneficum and did not display cross-reactivity with other microalgal species. The E-RCA-LFD assay was 100-fold more sensitive than conventional polymerase chain reaction (PCR) and had detection limits of 8.0 × 10¹ ag μL⁻¹ for the genomic DNA of K. veneficum and 5.0 × 10¹ ag μL⁻¹ (approximately 13 copies μL⁻¹) for recombinant plasmids containing the LSU rDNA D1–D2 regions of K. veneficum. Simulative tests indicated that the E-RCA-LFD assay demonstrated considerably higher sensitivity than conventional PCR and a detection limit of 0.01 cell mL⁻¹. The practicability of the E-RCA-LFD assay was confirmed through tests with field samples. In conclusion, the highly sensitive, specific, and facile E-RCA-LFD assay established in this work may enable the field monitoring of natural samples containing K. veneficum.