Recombinase polymerase amplification combined with lateral flow dipstick for the rapid detection of Prorocentrum donghaiense

Abstract

ABSTRACT Prorocentrum donghaiense is one of the most important harmful algal bloom (HAB)-forming species in the East China Sea. HABs caused by this alga has posed a considerable threat to marine ecosystems, fisheries, and human health. Therefore, exploring new methods for effectively detecting P. donghaiense is necessary. In this study, a novel method referred to as recombinase polymerase amplification combined with lateral flow dipstick (RPA−LFD), was developed for the detection of P. donghaiense. The internal transcribed sequence (ITS) of P. donghaiense was first obtained through PCR amplification, molecular cloning, and sequencing. Next, candidate RPA primers and LFD probes were designed through homologous sequence alignment analysis, and ITS was used as the target sequence. The optimal primers obtained through screening were used in establishing and optimizing the RPA−LFD detection system. Finally, the specificity of the system was verified, and its sensitivity and practicality were evaluated. Results showed that the RPA-LFD system had good specificity and did not cross-react with the other control algal species. The detection limit was 6.79 × 10−2 ng/μL for genomic DNA, 6.01 × 10 copies/μL for the ITS-containing recombinant plasmids, and 10 cells/mL for simulative water samples. The sensitivity of RPA−LFD was generally ten times that of conventional PCR. In conclusion, the proposed visual detection method for P. donghaiense is simple, rapid, sensitive, and applicable to field detection without complex instrumentation, and it is a promising tool for detecting P. donghaiense.