schliessen

Filtern

 

Bibliotheken

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... Full description

Journal Title: PLoS ONE 01 January 2018, Vol.13(5), p.e0196525
Main Author: Sining Han
Other Authors: Na Jiang , Qingyang Lv , Yumin Kan , Jianjun Hao , Jianqiang Li , Laixin Luo
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0196525
Zum Text:
SendSend as email Add to Book BagAdd to Book Bag
Staff View
recordid: doaj_soai_doaj_org_article_9d14dd66f06646c79bbdb43790f2cb7f
title: Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
format: Article
creator:
  • Sining Han
  • Na Jiang
  • Qingyang Lv
  • Yumin Kan
  • Jianjun Hao
  • Jianqiang Li
  • Laixin Luo
subjects:
  • Sciences (General)
ispartof: PLoS ONE, 01 January 2018, Vol.13(5), p.e0196525
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.sup.8 CFU mL.sup.-1) was induced by 50 [mu]M 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 [mu]M for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 10.sup.3 to 10.sup.7 CFU mL.sup.-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.
language: eng
source:
identifier: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0196525
fulltext: fulltext_linktorsrc
issn:
  • 1932-6203
  • 19326203
url: Link


@attributes
ID1159675814
RANK0.07
NO1
SEARCH_ENGINEprimo_central_multiple_fe
SEARCH_ENGINE_TYPEPrimo Central Search Engine
LOCALfalse
PrimoNMBib
record
control
sourcerecordidoai_doaj_org_article_9d14dd66f06646c79bbdb43790f2cb7f
sourceiddoaj_s
recordidTN_doaj_soai_doaj_org_article_9d14dd66f06646c79bbdb43790f2cb7f
sourcesystemPC
dbidDOA
pqid2034348381
galeid537218306
display
typearticle
titleDetection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
creatorSining Han ; Na Jiang ; Qingyang Lv ; Yumin Kan ; Jianjun Hao ; Jianqiang Li ; Laixin Luo
ispartofPLoS ONE, 01 January 2018, Vol.13(5), p.e0196525
identifierE-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0196525
subjectSciences (General)
languageeng
oafree_for_read
source
descriptionClavibacter 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.sup.8 CFU mL.sup.-1) was induced by 50 [mu]M 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 [mu]M for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 10.sup.3 to 10.sup.7 CFU mL.sup.-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.
version7
lds50peer_reviewed
links
openurl$$Topenurl_article
openurlfulltext$$Topenurlfull_article
linktorsrc$$Uhttps://doaj.org/article/9d14dd66f06646c79bbdb43790f2cb7f$$EView_full_text_in_DOAJ
search
creatorcontrib
0Sining Han
1Na Jiang
2Qingyang Lv
3Yumin Kan
4Jianjun Hao
5Jianqiang Li
6Laixin Luo
titleDetection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
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...

subjectSciences (General)
general
0English
1Public Library of Science (PLoS)
210.1371/journal.pone.0196525
3Directory of Open Access Journals (DOAJ)
sourceiddoaj_s
recordiddoaj_soai_doaj_org_article_9d14dd66f06646c79bbdb43790f2cb7f
issn
01932-6203
119326203
rsrctypearticle
creationdate2018
addtitlePLoS ONE
searchscope
0doaj_full
1doaj1
scope
0doaj_full
1doaj1
lsr45$$EView_full_text_in_DOAJ
tmp01Directory of Open Access Journals (DOAJ)
tmp02DOA
startdate20180101
enddate20180101
lsr40PLoS ONE, 01 January 2018, Vol.13 (5), p.e0196525
doi10.1371/journal.pone.0196525
citationpf e0196525 vol 13 issue 5
lsr30VSR-Enriched:[pages, description, pqid, galeid]
sort
titleDetection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
authorSining Han ; Na Jiang ; Qingyang Lv ; Yumin Kan ; Jianjun Hao ; Jianqiang Li ; Laixin Luo
creationdate20180101
lso0120180101
facets
frbrgroupid7684913714919616016
frbrtype5
newrecords20200226
languageeng
topicSciences (General)
collectionDirectory of Open Access Journals (DOAJ)
prefilterarticles
rsrctypearticles
creatorcontrib
0Sining Han
1Na Jiang
2Qingyang Lv
3Yumin Kan
4Jianjun Hao
5Jianqiang Li
6Laixin Luo
jtitlePLoS ONE
creationdate2018
toplevelpeer_reviewed
delivery
delcategoryRemote Search Resource
fulltextfulltext_linktorsrc
addata
au
0Sining Han
1Na Jiang
2Qingyang Lv
3Yumin Kan
4Jianjun Hao
5Jianqiang Li
6Laixin Luo
atitleDetection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
jtitlePLoS ONE
risdate20180101
volume13
issue5
spagee0196525
eissn1932-6203
formatjournal
genrearticle
ristypeJOUR
abstract

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...

pubPublic Library of Science (PLoS)
doi10.1371/journal.pone.0196525
urlhttps://doaj.org/article/9d14dd66f06646c79bbdb43790f2cb7f
lad01PLoS ONE
oafree_for_read
pagese0196525
date2018-01-01