Recombinase polymerase amplification combined with lateral-flow dipstick for rapid detection of Prorocentrum minimum

Abstract

The dinoflagellate Prorocentrum minimum is a common harmful algal bloom-forming microalgal species worldwide. Because of its harmfulness, accurate detection of P. minimum by convenient method has become a hotspot for field monitoring of this alga. The recently developed nucleic acid isothermal amplification technology is a promising tool for diagnosis and on-site detection of harmful microalgae. In this study, a novel assay based on isothermal amplification referred to recombinase polymerase amplification (RPA) combined with lateral-flow dipstick (LFD) was established for the detection of P. minimum. The specificity of the proposed RPA-LFD was validated by test with control algae. Various RPA conditions including amplification time, amplification temperature, and magnesium ion concentration in the amplification reaction were optimized. The RPA-LFD displayed a stable performance for the detection of P. minimum that is not disturbed by non-specific species. The optimal RPA-LFD could recognize the target algal species from water samples with a cell density of 10 cells mL−1 within 30 min, displaying a detection limit that was 10-fold lower than that of conventional PCR. The practicability of the proposed RPA-LFD was also confirmed by test with natural samples. In conclusion, the developed RPA-LFD is promising for rapid on-site detection of P. minimum in the future.