Development of a double-probe rolling circle amplification-lateral flow dipstick assay for rapid detection of...

Ensuring high-quality coastal waters is essential for maintaining healthy ecosystems, sustainable fisheries, and public health. At present, Amphidinium carterae poses a significant threat to many coastal nations. A novel technique, referred to as double probe rolling circle amplification-lateral flow dipstick (dpRCA-LFD), has been developed to detect A. carterae. This method employs the internal transcribed spacer (ITS) of A. carterae and the metallothionein-2 gene from Caenorhabditis elegans, a phylogenetically unrelated species, as a reference sequence. Specific probes and primers were designed based on these sequences. The dpRCA-LFD method yields results that are visually interpretable and requires no specialized equipment. The optimized reaction conditions were as follows: a circular ligation temperature of 61 °C, 10 cycles of circular ligation, an amplification temperature of 59 °C, and an amplification time of 50 min. Sensitivity testing demonstrated that dpRCA-LFD outperformed conventional PCR by two orders of magnitude, detecting genomic DNA concentrations ranging from 4.5 × 10¹ ng μL^-1 to 4.5 × 10^-4 ng μL^-1, and recombinant plasmids containing the ITS sequence at concentrations as low as 1.3 copies μL^-1. Both dpRCA and dpRCA-LFD were capable of detecting a single cell from simulated water samples, showing 100-fold better sensitivity than traditional PCR. In addition, dpRCA-LFD was capable of detecting target cells from natural water samples spiked with A. carterae. In summary, dpRCA-LFD offers high specificity, sensitivity, and easily interpretable results, rendering it a promising tool for monitoring A. carterae in aquatic environments.