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

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.

KEYWORDS:

• Prorocentrum donghaiense
• recombinase polymerase amplification
• lateral flow dipstick
• detection

Disclosure statement

No potential conflict of interest was reported by the author(s).

Compliance with ethical standards

All the authors would like to seriously state that: (a) the material used in this MS has not been published in whole or in part elsewhere; (b) the paper is not currently being considered for publication elsewhere; (c) all authors have been personally and actively involved in substantive work leading to the report, and will hold themselves jointly and individually responsible for its content; and (d) all relevant ethical safeguards have been met in this study.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.

Authors’ contributions

Conceived and designed the experiments: Chunyun Zhang and Guofu Chen; Performed the experiments: Yida Zhai and Runqi Li; Analyzed the data: Yuanyuan Wang. Contributed reagents/ materials/analysis tools: Fuguo Liu. Wrote the paper: Yida Zhai.

Consent to Publish

All the author's consent to publish all the data included in the MS.

Additional information

Funding

This study was founded by Shandong Provincial Natural Science Foundation, China (No.ZR2020MD081); the National Scientific Foundation of China (No. 31600309, 41476086); HIT Scientific Research Innovation Fund/the Fundamental Research Funds for the Central Universities (No. HIT.NSRIF.201702 and HIT.NSRIF.201709); and HIT Environment and Ecology Innovation Special Funds (No. HSCJ201622).