Detection of Phytophthora infestans by Loop-Mediated Isothermal Amplification, Real-Time LAMP, and Droplet Digital PCR.
Identifieur interne : 000250 ( Main/Exploration ); précédent : 000249; suivant : 000251Detection of Phytophthora infestans by Loop-Mediated Isothermal Amplification, Real-Time LAMP, and Droplet Digital PCR.
Auteurs : Jean B. Ristaino [États-Unis] ; Amanda C. Saville [États-Unis] ; Rajesh Paul [États-Unis] ; Donald C. Cooper [États-Unis] ; Qingshan Wei [États-Unis]Source :
- Plant disease [ 0191-2917 ] ; 2020.
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Abstract
Phytophthora infestans is the causal agent of potato late blight, a devastating disease of tomato and potato and a threat to global food security. Early detection and intervention is essential for effective management of the pathogen. We developed a loop-mediated isothermal amplification (LAMP) assay for P. infestans and compared this assay to conventional PCR, real-time LAMP, and droplet digital PCR for detection of P. infestans. The LAMP assay was specific for P. infestans on potato and tomato and did not amplify other potato- or tomato-infecting Phytophthora species or other fungal and bacterial pathogens that infect potato and tomato. The detection threshold for SYBR Green LAMP and real-time LAMP read with hydroxynaphthol blue and EvaGreen was 1 pg/µl. In contrast, detection by conventional PCR was 10 pg/µl. Droplet digital PCR had the lowest detection threshold (100 fg/µl). We adapted the LAMP assay using SYBR Green and a mobile reader (mReader) for use in the field. Detection limits were 584 fg/µl for SYBR Green LAMP read on the mReader, which was more sensitive than visualization with the human eye. The mobile platform records geospatial coordinates and data from positive pathogen detections can be directly uploaded to a cloud database. Data can then be integrated into disease surveillance networks. This system will be useful for real-time detection of P. infestans and will improve the timeliness of reports into surveillance systems such as USABlight or EuroBlight.
DOI: 10.1094/PDIS-06-19-1186-RE
PubMed: 31967506
Affiliations:
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Ristaino</name><affiliation wicri:level="2"><nlm:affiliation>Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Caroline du Nord</region></placeName><wicri:cityArea>Department of Entomology and Plant Pathology, North Carolina State University, Raleigh</wicri:cityArea></affiliation><affiliation wicri:level="2"><nlm:affiliation>Emerging Plant Disease and Global Food Security Cluster, North Carolina State University, Raleigh, NC 27695.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Caroline du Nord</region></placeName><wicri:cityArea>Emerging Plant Disease and Global Food Security Cluster, North Carolina State University, Raleigh</wicri:cityArea></affiliation></author><author><name sortKey="Saville, Amanda C" sort="Saville, Amanda C" uniqKey="Saville A" first="Amanda C" last="Saville">Amanda C. 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Cooper</name><affiliation wicri:level="2"><nlm:affiliation>Mobile Assay, Inc., Boulder, CO 80301.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Colorado</region></placeName><wicri:cityArea>Mobile Assay, Inc., Boulder</wicri:cityArea></affiliation></author><author><name sortKey="Wei, Qingshan" sort="Wei, Qingshan" uniqKey="Wei Q" first="Qingshan" last="Wei">Qingshan Wei</name><affiliation wicri:level="2"><nlm:affiliation>Emerging Plant Disease and Global Food Security Cluster, North Carolina State University, Raleigh, NC 27695.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Caroline du Nord</region></placeName><wicri:cityArea>Emerging Plant Disease and Global Food Security Cluster, North Carolina State University, Raleigh</wicri:cityArea></affiliation><affiliation wicri:level="2"><nlm:affiliation>Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Caroline du Nord</region></placeName><wicri:cityArea>Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh</wicri:cityArea></affiliation></author></analytic><series><title level="j">Plant disease</title><idno type="ISSN">0191-2917</idno><imprint><date when="2020" type="published">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Lycopersicon esculentum (MeSH)</term><term>Nucleic Acid Amplification Techniques (MeSH)</term><term>Phytophthora infestans (MeSH)</term><term>Polymerase Chain Reaction (MeSH)</term><term>Solanum tuberosum (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Lycopersicon esculentum (MeSH)</term><term>Phytophthora infestans (MeSH)</term><term>Réaction de polymérisation en chaîne (MeSH)</term><term>Solanum tuberosum (MeSH)</term><term>Techniques d'amplification d'acides nucléiques (MeSH)</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Lycopersicon esculentum</term><term>Nucleic Acid Amplification Techniques</term><term>Phytophthora infestans</term><term>Polymerase Chain Reaction</term><term>Solanum tuberosum</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Lycopersicon esculentum</term><term>Phytophthora infestans</term><term>Réaction de polymérisation en chaîne</term><term>Solanum tuberosum</term><term>Techniques d'amplification d'acides nucléiques</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><i>Phytophthora infestans</i> is the causal agent of potato late blight, a devastating disease of tomato and potato and a threat to global food security. Early detection and intervention is essential for effective management of the pathogen. We developed a loop-mediated isothermal amplification (LAMP) assay for <i>P. infestans</i> and compared this assay to conventional PCR, real-time LAMP, and droplet digital PCR for detection of <i>P. infestans</i>. The LAMP assay was specific for <i>P. infestans</i> on potato and tomato and did not amplify other potato- or tomato-infecting <i>Phytophthora</i> species or other fungal and bacterial pathogens that infect potato and tomato. The detection threshold for SYBR Green LAMP and real-time LAMP read with hydroxynaphthol blue and EvaGreen was 1 pg/µl. In contrast, detection by conventional PCR was 10 pg/µl. Droplet digital PCR had the lowest detection threshold (100 fg/µl). We adapted the LAMP assay using SYBR Green and a mobile reader (mReader) for use in the field. Detection limits were 584 fg/µl for SYBR Green LAMP read on the mReader, which was more sensitive than visualization with the human eye. The mobile platform records geospatial coordinates and data from positive pathogen detections can be directly uploaded to a cloud database. Data can then be integrated into disease surveillance networks. This system will be useful for real-time detection of <i>P. infestans</i> and will improve the timeliness of reports into surveillance systems such as USABlight or EuroBlight.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" IndexingMethod="Automated" Owner="NLM"><PMID Version="1">31967506</PMID><DateCompleted><Year>2020</Year><Month>03</Month><Day>05</Day></DateCompleted><DateRevised><Year>2020</Year><Month>03</Month><Day>05</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0191-2917</ISSN><JournalIssue CitedMedium="Print"><Volume>104</Volume><Issue>3</Issue><PubDate><Year>2020</Year><Month>Mar</Month></PubDate></JournalIssue><Title>Plant disease</Title><ISOAbbreviation>Plant Dis</ISOAbbreviation></Journal><ArticleTitle>Detection of <i>Phytophthora infestans</i> by Loop-Mediated Isothermal Amplification, Real-Time LAMP, and Droplet Digital PCR.</ArticleTitle><Pagination><MedlinePgn>708-716</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1094/PDIS-06-19-1186-RE</ELocationID><Abstract><AbstractText><i>Phytophthora infestans</i> is the causal agent of potato late blight, a devastating disease of tomato and potato and a threat to global food security. Early detection and intervention is essential for effective management of the pathogen. We developed a loop-mediated isothermal amplification (LAMP) assay for <i>P. infestans</i> and compared this assay to conventional PCR, real-time LAMP, and droplet digital PCR for detection of <i>P. infestans</i>. The LAMP assay was specific for <i>P. infestans</i> on potato and tomato and did not amplify other potato- or tomato-infecting <i>Phytophthora</i> species or other fungal and bacterial pathogens that infect potato and tomato. The detection threshold for SYBR Green LAMP and real-time LAMP read with hydroxynaphthol blue and EvaGreen was 1 pg/µl. In contrast, detection by conventional PCR was 10 pg/µl. Droplet digital PCR had the lowest detection threshold (100 fg/µl). We adapted the LAMP assay using SYBR Green and a mobile reader (mReader) for use in the field. Detection limits were 584 fg/µl for SYBR Green LAMP read on the mReader, which was more sensitive than visualization with the human eye. The mobile platform records geospatial coordinates and data from positive pathogen detections can be directly uploaded to a cloud database. Data can then be integrated into disease surveillance networks. This system will be useful for real-time detection of <i>P. infestans</i> and will improve the timeliness of reports into surveillance systems such as USABlight or EuroBlight.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Ristaino</LastName><ForeName>Jean B</ForeName><Initials>JB</Initials><Identifier Source="ORCID">http://orcid.org/0000-0002-9458-0514</Identifier><AffiliationInfo><Affiliation>Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Emerging Plant Disease and Global Food Security Cluster, North Carolina State University, Raleigh, NC 27695.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Saville</LastName><ForeName>Amanda C</ForeName><Initials>AC</Initials><AffiliationInfo><Affiliation>Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Paul</LastName><ForeName>Rajesh</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Cooper</LastName><ForeName>Donald C</ForeName><Initials>DC</Initials><AffiliationInfo><Affiliation>Mobile Assay, Inc., Boulder, CO 80301.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Wei</LastName><ForeName>Qingshan</ForeName><Initials>Q</Initials><AffiliationInfo><Affiliation>Emerging Plant Disease and Global Food Security Cluster, North Carolina State University, Raleigh, NC 27695.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2020</Year><Month>01</Month><Day>22</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Plant Dis</MedlineTA><NlmUniqueID>9882809</NlmUniqueID><ISSNLinking>0191-2917</ISSNLinking></MedlineJournalInfo><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D018551" MajorTopicYN="Y">Lycopersicon esculentum</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D021141" MajorTopicYN="N">Nucleic Acid Amplification Techniques</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D055750" MajorTopicYN="Y">Phytophthora infestans</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D016133" MajorTopicYN="N">Polymerase Chain Reaction</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011198" MajorTopicYN="Y">Solanum tuberosum</DescriptorName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Phytophthora infestans</Keyword><Keyword MajorTopicYN="N">oomycetes</Keyword><Keyword MajorTopicYN="N">pathogen detection</Keyword><Keyword MajorTopicYN="N">vegetables</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2020</Year><Month>1</Month><Day>23</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2020</Year><Month>3</Month><Day>7</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2020</Year><Month>1</Month><Day>23</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">31967506</ArticleId><ArticleId IdType="doi">10.1094/PDIS-06-19-1186-RE</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country><region><li>Caroline du Nord</li><li>Colorado</li></region></list><tree><country name="États-Unis"><region name="Caroline du Nord"><name sortKey="Ristaino, Jean B" sort="Ristaino, Jean B" uniqKey="Ristaino J" first="Jean B" last="Ristaino">Jean B. Ristaino</name></region><name sortKey="Cooper, Donald C" sort="Cooper, Donald C" uniqKey="Cooper D" first="Donald C" last="Cooper">Donald C. Cooper</name><name sortKey="Paul, Rajesh" sort="Paul, Rajesh" uniqKey="Paul R" first="Rajesh" last="Paul">Rajesh Paul</name><name sortKey="Ristaino, Jean B" sort="Ristaino, Jean B" uniqKey="Ristaino J" first="Jean B" last="Ristaino">Jean B. Ristaino</name><name sortKey="Saville, Amanda C" sort="Saville, Amanda C" uniqKey="Saville A" first="Amanda C" last="Saville">Amanda C. Saville</name><name sortKey="Wei, Qingshan" sort="Wei, Qingshan" uniqKey="Wei Q" first="Qingshan" last="Wei">Qingshan Wei</name><name sortKey="Wei, Qingshan" sort="Wei, Qingshan" uniqKey="Wei Q" first="Qingshan" last="Wei">Qingshan Wei</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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