Cargando…

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

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 c...

Descripción completa

Detalles Bibliográficos
Autores principales: Dai, Tingting, Hu, Tao, Yang, Xiao, Shen, Danyu, Jiao, Binbin, Tian, Wen, Xu, Yue
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6870529/
https://www.ncbi.nlm.nih.gov/pubmed/31763074
http://dx.doi.org/10.7717/peerj.8083
_version_ 1783472401351180288
author Dai, Tingting
Hu, Tao
Yang, Xiao
Shen, Danyu
Jiao, Binbin
Tian, Wen
Xu, Yue
author_facet Dai, Tingting
Hu, Tao
Yang, Xiao
Shen, Danyu
Jiao, Binbin
Tian, Wen
Xu, Yue
author_sort Dai, Tingting
collection PubMed
description 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.
format Online
Article
Text
id pubmed-6870529
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher PeerJ Inc.
record_format MEDLINE/PubMed
spelling pubmed-68705292019-11-23 A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis Dai, Tingting Hu, Tao Yang, Xiao Shen, Danyu Jiao, Binbin Tian, Wen Xu, Yue PeerJ Microbiology 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. PeerJ Inc. 2019-11-18 /pmc/articles/PMC6870529/ /pubmed/31763074 http://dx.doi.org/10.7717/peerj.8083 Text en © 2019 Dai et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Microbiology
Dai, Tingting
Hu, Tao
Yang, Xiao
Shen, Danyu
Jiao, Binbin
Tian, Wen
Xu, Yue
A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis
title A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis
title_full A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis
title_fullStr A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis
title_full_unstemmed A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis
title_short A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis
title_sort recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in china, phytophthora hibernalis
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6870529/
https://www.ncbi.nlm.nih.gov/pubmed/31763074
http://dx.doi.org/10.7717/peerj.8083
work_keys_str_mv AT daitingting arecombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT hutao arecombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT yangxiao arecombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT shendanyu arecombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT jiaobinbin arecombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT tianwen arecombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT xuyue arecombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT daitingting recombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT hutao recombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT yangxiao recombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT shendanyu recombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT jiaobinbin recombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT tianwen recombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis
AT xuyue recombinasepolymeraseamplificationlateralflowdipstickassayforrapiddetectionofthequarantinecitruspathogeninchinaphytophthorahibernalis