A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis

PeerJ. 2019 Nov 18:7:e8083. doi: 10.7717/peerj.8083. eCollection 2019.

Abstract

Phytophthora hibernalis, the causal agent of brown rot of citrus fruit, is an important worldwide pathogen and a quarantine pest in China. Current diagnosis of the disease relies on disease symptoms, pathogen isolation and identification by DNA sequencing. However, symptoms caused by P. hibernalis can be confused with those by other Phytophthora and fungal species. Moreover, pathogen isolation, PCR amplification and sequencing are time-consuming. In this study, a rapid assay including 20-min recombinase polymerase amplification targeting the Ypt1 gene and 5-min visualization using lateral flow dipsticks was developed for detecting P. hibernalis. This assay was able to detect 0.2 ng of P. hibernalis genomic DNA in a 50-µL reaction system. It was specific to P. hibernalis without detection of other tested species including P. citrophthora, P. nicotianae, P. palmivora and P. syringae, four other important citrus pathogens. Using this assay, P. hibernalis was also detected from artificially inoculated orange fruits. Results in this study indicated that this assay has the potential application to detect P. hibernalis at diagnostic laboratories and plant quarantine departments of customs, especially under time- and resource-limited conditions.

Keywords: Exclusivity; Inclusivity; Isothermal diagnostic assay; Oomycetes; Plant destroyers; RPA–LFD.

Grants and funding

This work was supported by the National Natural Science Foundation of China (31500526), China Postdoctoral Science Foundation (2016T790467), Overseas Research and Study Project of Excellent Young and Middle-aged Teachers and Principals in Colleges and Universities of Jiangsu Province of 2018, the Priority Academic Program Development of Jiangsu Higher Education Institutions, Special Fund for Agro-scientific Research in the Public Interest (201503112), Jiangsu Basic Research Program (Natural Science Foundation) Project (BK 20191389) and Jiangsu University Natural Science Research Surface Project (19KJB220003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.