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Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (V...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933903/ https://www.ncbi.nlm.nih.gov/pubmed/29723290 http://dx.doi.org/10.1371/journal.pone.0196525 |
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author | Han, Sining Jiang, Na Lv, Qingyang Kan, Yumin Hao, Jianjun Li, Jianqiang Luo, Laixin |
author_facet | Han, Sining Jiang, Na Lv, Qingyang Kan, Yumin Hao, Jianjun Li, Jianqiang Luo, Laixin |
author_sort | Han, Sining |
collection | PubMed |
description | Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (10(8) CFU mL(-1)) was induced by 50 μM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 μM for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 10(3) to 10(7) CFU mL(-1). The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set. |
format | Online Article Text |
id | pubmed-5933903 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-59339032018-05-18 Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR Han, Sining Jiang, Na Lv, Qingyang Kan, Yumin Hao, Jianjun Li, Jianqiang Luo, Laixin PLoS One Research Article Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (10(8) CFU mL(-1)) was induced by 50 μM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 μM for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 10(3) to 10(7) CFU mL(-1). The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set. Public Library of Science 2018-05-03 /pmc/articles/PMC5933903/ /pubmed/29723290 http://dx.doi.org/10.1371/journal.pone.0196525 Text en © 2018 Han et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Han, Sining Jiang, Na Lv, Qingyang Kan, Yumin Hao, Jianjun Li, Jianqiang Luo, Laixin Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR |
title | Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR |
title_full | Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR |
title_fullStr | Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR |
title_full_unstemmed | Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR |
title_short | Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR |
title_sort | detection of clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qpcr |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933903/ https://www.ncbi.nlm.nih.gov/pubmed/29723290 http://dx.doi.org/10.1371/journal.pone.0196525 |
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