Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated...

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Main Authors:	Yao, Xiefeng, Li, Pingfang, Xu, Jinghua, Zhang, Man, Ren, Runsheng, Liu, Guang, Yang, Xingping
Format:	Online
Language:	English
Published:	Frontiers Media S.A. 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003822/

id	pubmed-5003822
recordtype	oai_dc
spelling	pubmed-50038222016-09-13 Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay Yao, Xiefeng Li, Pingfang Xu, Jinghua Zhang, Man Ren, Runsheng Liu, Guang Yang, Xingping Microbiology Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics. Frontiers Media S.A. 2016-08-30 /pmc/articles/PMC5003822/ /pubmed/27625648 http://dx.doi.org/10.3389/fmicb.2016.01372 Text en Copyright © 2016 Yao, Li, Xu, Zhang, Ren, Liu and Yang. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Yao, Xiefeng Li, Pingfang Xu, Jinghua Zhang, Man Ren, Runsheng Liu, Guang Yang, Xingping
spellingShingle	Yao, Xiefeng Li, Pingfang Xu, Jinghua Zhang, Man Ren, Runsheng Liu, Guang Yang, Xingping Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay
author_facet	Yao, Xiefeng Li, Pingfang Xu, Jinghua Zhang, Man Ren, Runsheng Liu, Guang Yang, Xingping
author_sort	Yao, Xiefeng
title	Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay
title_short	Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay
title_full	Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay
title_fullStr	Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay
title_full_unstemmed	Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay
title_sort	rapid and sensitive detection of didymella bryoniae by visual loop-mediated isothermal amplification assay
description	Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.
publisher	Frontiers Media S.A.
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003822/
_version_	1613638453456535552

Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

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