A New Application Using a Chromogenic Assay in a Plant Pathogen DNA Macroarray Detection System.
Identifieur interne : 001606 ( Main/Exploration ); précédent : 001605; suivant : 001607A New Application Using a Chromogenic Assay in a Plant Pathogen DNA Macroarray Detection System.
Auteurs : Mui-Yun Wong [Malaisie] ; Christine D. Smart [États-Unis]Source :
- Plant disease [ 0191-2917 ] ; 2012.
Abstract
A DNA macroarray was previously developed to detect major fungal and oomycete pathogens of solanaceous crops. To provide a convenient alternative for researchers with no access to X-ray film-developing facilities, specific CCD cameras or Chemidoc XRS systems, a chromogenic detection method with sensitivity comparable with chemiluminescent detection, has been developed. A fungal (Stemphylium solani) and an oomycete (Phytophthora capsici) pathogen were used to develop the protocol using digoxigenin (DIG)-labeled targets. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, and ITS2, was used as the target gene and polymerase chain reaction amplified as in the previous protocol. Various amounts of species-specific oligonucleotides on the array, quantities of DIG-labeled ITS amplicon, and hybridization temperatures were tested. The optimal conditions for hybridization were 55°C for 2 h using at least 10 pmol of each species-specific oligonucleotide and labeled target at 10 ng/ml of hybridization buffer. Incubation of the hybridized array with anti-DIG conjugated alkaline phosphatase substrates, NBT/BCIP, produced visible target signals between 1 and 3 h compared with 1 h in chemiluminescent detection. Samples from pure cultures, soil, and artificially inoculated plants were also used to compare the detection using chemiluminescent and chromogenic methods. Chromogenic detection was shown to yield similar results compared with chemiluminescent detection in regard to signal specificity, duration of hybridization between the array and targets, and cost, though it takes 1 to 2 h longer for the visualization process, thus providing a convenient alternative for researchers who lack darkroom facilities. To our knowledge, this is the first report of DNA macroarray detection of plant pathogens using a chromogenic method.
DOI: 10.1094/PDIS-07-11-0593-SR
PubMed: 30727148
Affiliations:
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Smart</name><affiliation wicri:level="2"><nlm:affiliation>Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva, NY 14456.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">État de New York</region></placeName><wicri:cityArea>Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva</wicri:cityArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2012">2012</date><idno type="RBID">pubmed:30727148</idno><idno type="pmid">30727148</idno><idno type="doi">10.1094/PDIS-07-11-0593-SR</idno><idno type="wicri:Area/Main/Corpus">001451</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">001451</idno><idno type="wicri:Area/Main/Curation">001451</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">001451</idno><idno type="wicri:Area/Main/Exploration">001451</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">A New Application Using a Chromogenic Assay in a Plant Pathogen DNA Macroarray Detection System.</title><author><name sortKey="Wong, Mui Yun" sort="Wong, Mui Yun" uniqKey="Wong M" first="Mui-Yun" last="Wong">Mui-Yun Wong</name><affiliation wicri:level="1"><nlm:affiliation>Department of Plant Protection, Faculty of Agriculture, and Institute of Tropical Agriculture, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia.</nlm:affiliation><country xml:lang="fr">Malaisie</country><wicri:regionArea>Department of Plant Protection, Faculty of Agriculture, and Institute of Tropical Agriculture, Universiti Putra Malaysia, Serdang, Selangor 43400</wicri:regionArea><wicri:noRegion>Selangor 43400</wicri:noRegion></affiliation></author><author><name sortKey="Smart, Christine D" sort="Smart, Christine D" uniqKey="Smart C" first="Christine D" last="Smart">Christine D. Smart</name><affiliation wicri:level="2"><nlm:affiliation>Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva, NY 14456.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">État de New York</region></placeName><wicri:cityArea>Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva</wicri:cityArea></affiliation></author></analytic><series><title level="j">Plant disease</title><idno type="ISSN">0191-2917</idno><imprint><date when="2012" type="published">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">A DNA macroarray was previously developed to detect major fungal and oomycete pathogens of solanaceous crops. To provide a convenient alternative for researchers with no access to X-ray film-developing facilities, specific CCD cameras or Chemidoc XRS systems, a chromogenic detection method with sensitivity comparable with chemiluminescent detection, has been developed. A fungal (Stemphylium solani) and an oomycete (Phytophthora capsici) pathogen were used to develop the protocol using digoxigenin (DIG)-labeled targets. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, and ITS2, was used as the target gene and polymerase chain reaction amplified as in the previous protocol. Various amounts of species-specific oligonucleotides on the array, quantities of DIG-labeled ITS amplicon, and hybridization temperatures were tested. The optimal conditions for hybridization were 55°C for 2 h using at least 10 pmol of each species-specific oligonucleotide and labeled target at 10 ng/ml of hybridization buffer. Incubation of the hybridized array with anti-DIG conjugated alkaline phosphatase substrates, NBT/BCIP, produced visible target signals between 1 and 3 h compared with 1 h in chemiluminescent detection. Samples from pure cultures, soil, and artificially inoculated plants were also used to compare the detection using chemiluminescent and chromogenic methods. Chromogenic detection was shown to yield similar results compared with chemiluminescent detection in regard to signal specificity, duration of hybridization between the array and targets, and cost, though it takes 1 to 2 h longer for the visualization process, thus providing a convenient alternative for researchers who lack darkroom facilities. To our knowledge, this is the first report of DNA macroarray detection of plant pathogens using a chromogenic method.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">30727148</PMID><DateRevised><Year>2019</Year><Month>11</Month><Day>20</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0191-2917</ISSN><JournalIssue CitedMedium="Print"><Volume>96</Volume><Issue>9</Issue><PubDate><Year>2012</Year><Month>Sep</Month></PubDate></JournalIssue><Title>Plant disease</Title><ISOAbbreviation>Plant Dis</ISOAbbreviation></Journal><ArticleTitle>A New Application Using a Chromogenic Assay in a Plant Pathogen DNA Macroarray Detection System.</ArticleTitle><Pagination><MedlinePgn>1365-1371</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1094/PDIS-07-11-0593-SR</ELocationID><Abstract><AbstractText>A DNA macroarray was previously developed to detect major fungal and oomycete pathogens of solanaceous crops. To provide a convenient alternative for researchers with no access to X-ray film-developing facilities, specific CCD cameras or Chemidoc XRS systems, a chromogenic detection method with sensitivity comparable with chemiluminescent detection, has been developed. A fungal (Stemphylium solani) and an oomycete (Phytophthora capsici) pathogen were used to develop the protocol using digoxigenin (DIG)-labeled targets. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, and ITS2, was used as the target gene and polymerase chain reaction amplified as in the previous protocol. Various amounts of species-specific oligonucleotides on the array, quantities of DIG-labeled ITS amplicon, and hybridization temperatures were tested. The optimal conditions for hybridization were 55°C for 2 h using at least 10 pmol of each species-specific oligonucleotide and labeled target at 10 ng/ml of hybridization buffer. Incubation of the hybridized array with anti-DIG conjugated alkaline phosphatase substrates, NBT/BCIP, produced visible target signals between 1 and 3 h compared with 1 h in chemiluminescent detection. Samples from pure cultures, soil, and artificially inoculated plants were also used to compare the detection using chemiluminescent and chromogenic methods. Chromogenic detection was shown to yield similar results compared with chemiluminescent detection in regard to signal specificity, duration of hybridization between the array and targets, and cost, though it takes 1 to 2 h longer for the visualization process, thus providing a convenient alternative for researchers who lack darkroom facilities. To our knowledge, this is the first report of DNA macroarray detection of plant pathogens using a chromogenic method.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Wong</LastName><ForeName>Mui-Yun</ForeName><Initials>MY</Initials><AffiliationInfo><Affiliation>Department of Plant Protection, Faculty of Agriculture, and Institute of Tropical Agriculture, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Smart</LastName><ForeName>Christine D</ForeName><Initials>CD</Initials><AffiliationInfo><Affiliation>Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva, NY 14456.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Plant Dis</MedlineTA><NlmUniqueID>9882809</NlmUniqueID><ISSNLinking>0191-2917</ISSNLinking></MedlineJournalInfo></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="entrez"><Year>2019</Year><Month>2</Month><Day>8</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2012</Year><Month>9</Month><Day>1</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2012</Year><Month>9</Month><Day>1</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">30727148</ArticleId><ArticleId IdType="doi">10.1094/PDIS-07-11-0593-SR</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Malaisie</li><li>États-Unis</li></country><region><li>État de New York</li></region></list><tree><country name="Malaisie"><noRegion><name sortKey="Wong, Mui Yun" sort="Wong, Mui Yun" uniqKey="Wong M" first="Mui-Yun" last="Wong">Mui-Yun Wong</name></noRegion></country><country name="États-Unis"><region name="État de New York"><name sortKey="Smart, Christine D" sort="Smart, Christine D" uniqKey="Smart C" first="Christine D" last="Smart">Christine D. 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