Cargando…

A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac

Bacterial soft rot caused by Pectobacterium species is a serious disease in konjac (Amorphophallus konjac), a healthy source of starch particularly in East Asia. An effective diagnostic method is crucial to control the disease and reduce losses in konjac production. In this study, we evaluated a loo...

Descripción completa

Detalles Bibliográficos
Autores principales: Sun, Miaomiao, Liu, Hao, Huang, Junbin, Peng, Jinbo, Fei, Fuhua, Zhang, Ya, Hsiang, Tom, Zheng, Lu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514923/
https://www.ncbi.nlm.nih.gov/pubmed/31010197
http://dx.doi.org/10.3390/ijms20081937
_version_ 1783417973009022976
author Sun, Miaomiao
Liu, Hao
Huang, Junbin
Peng, Jinbo
Fei, Fuhua
Zhang, Ya
Hsiang, Tom
Zheng, Lu
author_facet Sun, Miaomiao
Liu, Hao
Huang, Junbin
Peng, Jinbo
Fei, Fuhua
Zhang, Ya
Hsiang, Tom
Zheng, Lu
author_sort Sun, Miaomiao
collection PubMed
description Bacterial soft rot caused by Pectobacterium species is a serious disease in konjac (Amorphophallus konjac), a healthy source of starch particularly in East Asia. An effective diagnostic method is crucial to control the disease and reduce losses in konjac production. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay with a specific primer set for the rapid and accurate detection of P. aroidearum. A comparative genomics approach was used to identify the specific genes suitable for the design of LAMP primers. The candidate target genes were determined through a first-round comparison with a 50-genome nucleotide database, and subjected to a second-round screening with the GenBank NR database. As a result, nine specific genes of P. aroidearum were selected for LAMP primer design. After screening of the primers, the primer set 1675-1 was chosen for LAMP detection owing to its high specificity and sensitivity. The LAMP assay could detect the presence of P. aroidearum genomic DNA at a concentration as low as 50 fg and 1.2 × 10(4) CFU/g artificially infected soil within 40 min at 65 °C. Subsequently, this primer set was successfully used to specifically detect P. aroidearum in naturally infected and non-symptomatic plant samples or soil samples from the field. This study indicates that a comparative genomic approach may facilitate the development of highly specific primers for LAMP assays, and a LAMP diagnostic assay with the specific primer set 1675-1 should contribute to the rapid and accurate detection of soft-rot disease in konjac at an early stage.
format Online
Article
Text
id pubmed-6514923
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-65149232019-05-30 A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac Sun, Miaomiao Liu, Hao Huang, Junbin Peng, Jinbo Fei, Fuhua Zhang, Ya Hsiang, Tom Zheng, Lu Int J Mol Sci Article Bacterial soft rot caused by Pectobacterium species is a serious disease in konjac (Amorphophallus konjac), a healthy source of starch particularly in East Asia. An effective diagnostic method is crucial to control the disease and reduce losses in konjac production. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay with a specific primer set for the rapid and accurate detection of P. aroidearum. A comparative genomics approach was used to identify the specific genes suitable for the design of LAMP primers. The candidate target genes were determined through a first-round comparison with a 50-genome nucleotide database, and subjected to a second-round screening with the GenBank NR database. As a result, nine specific genes of P. aroidearum were selected for LAMP primer design. After screening of the primers, the primer set 1675-1 was chosen for LAMP detection owing to its high specificity and sensitivity. The LAMP assay could detect the presence of P. aroidearum genomic DNA at a concentration as low as 50 fg and 1.2 × 10(4) CFU/g artificially infected soil within 40 min at 65 °C. Subsequently, this primer set was successfully used to specifically detect P. aroidearum in naturally infected and non-symptomatic plant samples or soil samples from the field. This study indicates that a comparative genomic approach may facilitate the development of highly specific primers for LAMP assays, and a LAMP diagnostic assay with the specific primer set 1675-1 should contribute to the rapid and accurate detection of soft-rot disease in konjac at an early stage. MDPI 2019-04-19 /pmc/articles/PMC6514923/ /pubmed/31010197 http://dx.doi.org/10.3390/ijms20081937 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sun, Miaomiao
Liu, Hao
Huang, Junbin
Peng, Jinbo
Fei, Fuhua
Zhang, Ya
Hsiang, Tom
Zheng, Lu
A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac
title A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac
title_full A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac
title_fullStr A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac
title_full_unstemmed A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac
title_short A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac
title_sort loop-mediated isothermal amplification assay for rapid detection of pectobacterium aroidearum that causes soft rot in konjac
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514923/
https://www.ncbi.nlm.nih.gov/pubmed/31010197
http://dx.doi.org/10.3390/ijms20081937
work_keys_str_mv AT sunmiaomiao aloopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT liuhao aloopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT huangjunbin aloopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT pengjinbo aloopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT feifuhua aloopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT zhangya aloopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT hsiangtom aloopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT zhenglu aloopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT sunmiaomiao loopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT liuhao loopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT huangjunbin loopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT pengjinbo loopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT feifuhua loopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT zhangya loopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT hsiangtom loopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac
AT zhenglu loopmediatedisothermalamplificationassayforrapiddetectionofpectobacteriumaroidearumthatcausessoftrotinkonjac