Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species.
Identifieur interne : 000011 ( Main/Exploration ); précédent : 000010; suivant : 000012Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species.
Auteurs : Austin G. Mccoy [États-Unis] ; Timothy D. Miles [États-Unis] ; Guillaume J. Bilodeau [Canada] ; Patrick Woods [États-Unis] ; Cheryl Blomquist [États-Unis] ; Frank N. Martin [États-Unis] ; Martin I. Chilvers [États-Unis]Source :
- Plants (Basel, Switzerland) [ 2223-7747 ] ; 2020.
Abstract
Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available in liquid form more recently-both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated Phytophthora genus-specific RPA assay, lyophilized with primers and probes included with the RPA reagents. The preformulated lyophilized Phytophthora RPA assay was compared with a quantitative polymerase chain reaction (qPCR) assay and commercially available RPA kits using three qPCR platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of Phytophthora-infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without added plant extract. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the consistency and uniformity of pathogen detection with preformulated RPA kits for Phytophthora detection when conducted by different labs using different instruments for measuring results.
DOI: 10.3390/plants9040466
PubMed: 32272704
PubMed Central: PMC7238109
Affiliations:
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Mccoy</name><affiliation wicri:level="4"><nlm:affiliation>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824</wicri:regionArea><placeName><region type="state">Michigan</region><settlement type="city">East Lansing</settlement></placeName><orgName type="university">Université d'État du Michigan</orgName></affiliation></author><author><name sortKey="Miles, Timothy D" sort="Miles, Timothy D" uniqKey="Miles T" first="Timothy D" last="Miles">Timothy D. Miles</name><affiliation wicri:level="4"><nlm:affiliation>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824</wicri:regionArea><placeName><region type="state">Michigan</region><settlement type="city">East Lansing</settlement></placeName><orgName type="university">Université d'État du Michigan</orgName></affiliation></author><author><name sortKey="Bilodeau, Guillaume J" sort="Bilodeau, Guillaume J" uniqKey="Bilodeau G" first="Guillaume J" last="Bilodeau">Guillaume J. Bilodeau</name><affiliation wicri:level="1"><nlm:affiliation>Ottawa Plant Laboratory, Canadian Food Inspection Agency, 3851 Fallowfield Road, Ottawa, ON K2H 8P9, Canada.</nlm:affiliation><country xml:lang="fr">Canada</country><wicri:regionArea>Ottawa Plant Laboratory, Canadian Food Inspection Agency, 3851 Fallowfield Road, Ottawa, ON K2H 8P9</wicri:regionArea><wicri:noRegion>ON K2H 8P9</wicri:noRegion></affiliation></author><author><name sortKey="Woods, Patrick" sort="Woods, Patrick" uniqKey="Woods P" first="Patrick" last="Woods">Patrick Woods</name><affiliation wicri:level="2"><nlm:affiliation>Plant Pest Diagnostics Branch, California Department of Food and Agriculture, Sacramento, CA 95832, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Plant Pest Diagnostics Branch, California Department of Food and Agriculture, Sacramento, CA 95832</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Blomquist, Cheryl" sort="Blomquist, Cheryl" uniqKey="Blomquist C" first="Cheryl" last="Blomquist">Cheryl Blomquist</name><affiliation wicri:level="2"><nlm:affiliation>Plant Pest Diagnostics Branch, California Department of Food and Agriculture, Sacramento, CA 95832, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Plant Pest Diagnostics Branch, California Department of Food and Agriculture, Sacramento, CA 95832</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Martin, Frank N" sort="Martin, Frank N" uniqKey="Martin F" first="Frank N" last="Martin">Frank N. Martin</name><affiliation wicri:level="2"><nlm:affiliation>Crop Improvement and Protection Research Unit, U.S. Department of Agriculture Agricultural Research Service, Salinas, CA 93905, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Crop Improvement and Protection Research Unit, U.S. Department of Agriculture Agricultural Research Service, Salinas, CA 93905</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Chilvers, Martin I" sort="Chilvers, Martin I" uniqKey="Chilvers M" first="Martin I" last="Chilvers">Martin I. Chilvers</name><affiliation wicri:level="4"><nlm:affiliation>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824</wicri:regionArea><placeName><region type="state">Michigan</region><settlement type="city">East Lansing</settlement></placeName><orgName type="university">Université d'État du Michigan</orgName></affiliation></author></analytic><series><title level="j">Plants (Basel, Switzerland)</title><idno type="ISSN">2223-7747</idno><imprint><date when="2020" type="published">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available in liquid form more recently-both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated <i>Phytophthora</i> genus-specific RPA assay, lyophilized with primers and probes included with the RPA reagents. The preformulated lyophilized <i>Phytophthora</i> RPA assay was compared with a quantitative polymerase chain reaction (qPCR) assay and commercially available RPA kits using three qPCR platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of <i>Phytophthora</i>-infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without added plant extract. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the consistency and uniformity of pathogen detection with preformulated RPA kits for <i>Phytophthora</i> detection when conducted by different labs using different instruments for measuring results.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">32272704</PMID><DateRevised><Year>2020</Year><Month>09</Month><Day>28</Day></DateRevised><Article PubModel="Electronic"><Journal><ISSN IssnType="Print">2223-7747</ISSN><JournalIssue CitedMedium="Print"><Volume>9</Volume><Issue>4</Issue><PubDate><Year>2020</Year><Month>Apr</Month><Day>07</Day></PubDate></JournalIssue><Title>Plants (Basel, Switzerland)</Title><ISOAbbreviation>Plants (Basel)</ISOAbbreviation></Journal><ArticleTitle>Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for <i>Phytophthora</i> Species.</ArticleTitle><ELocationID EIdType="pii" ValidYN="Y">E466</ELocationID><ELocationID EIdType="doi" ValidYN="Y">10.3390/plants9040466</ELocationID><Abstract><AbstractText>Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available in liquid form more recently-both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated <i>Phytophthora</i> genus-specific RPA assay, lyophilized with primers and probes included with the RPA reagents. The preformulated lyophilized <i>Phytophthora</i> RPA assay was compared with a quantitative polymerase chain reaction (qPCR) assay and commercially available RPA kits using three qPCR platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of <i>Phytophthora</i>-infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without added plant extract. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the consistency and uniformity of pathogen detection with preformulated RPA kits for <i>Phytophthora</i> detection when conducted by different labs using different instruments for measuring results.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>McCoy</LastName><ForeName>Austin G</ForeName><Initials>AG</Initials><Identifier Source="ORCID">0000-0003-2483-4184</Identifier><AffiliationInfo><Affiliation>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Miles</LastName><ForeName>Timothy D</ForeName><Initials>TD</Initials><AffiliationInfo><Affiliation>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Bilodeau</LastName><ForeName>Guillaume J</ForeName><Initials>GJ</Initials><Identifier Source="ORCID">0000-0003-1766-229X</Identifier><AffiliationInfo><Affiliation>Ottawa Plant Laboratory, Canadian Food Inspection Agency, 3851 Fallowfield Road, Ottawa, ON K2H 8P9, Canada.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Woods</LastName><ForeName>Patrick</ForeName><Initials>P</Initials><AffiliationInfo><Affiliation>Plant Pest Diagnostics Branch, California Department of Food and Agriculture, Sacramento, CA 95832, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Blomquist</LastName><ForeName>Cheryl</ForeName><Initials>C</Initials><AffiliationInfo><Affiliation>Plant Pest Diagnostics Branch, California Department of Food and Agriculture, Sacramento, CA 95832, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Martin</LastName><ForeName>Frank N</ForeName><Initials>FN</Initials><AffiliationInfo><Affiliation>Crop Improvement and Protection Research Unit, U.S. Department of Agriculture Agricultural Research Service, Salinas, CA 93905, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Chilvers</LastName><ForeName>Martin I</ForeName><Initials>MI</Initials><AffiliationInfo><Affiliation>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2020</Year><Month>04</Month><Day>07</Day></ArticleDate></Article><MedlineJournalInfo><Country>Switzerland</Country><MedlineTA>Plants (Basel)</MedlineTA><NlmUniqueID>101596181</NlmUniqueID><ISSNLinking>2223-7747</ISSNLinking></MedlineJournalInfo><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">diagnostics</Keyword><Keyword MajorTopicYN="N">isothermal amplification</Keyword><Keyword MajorTopicYN="N">oomycetes</Keyword><Keyword MajorTopicYN="N">point of care diagnostics</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2020</Year><Month>02</Month><Day>22</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2020</Year><Month>03</Month><Day>27</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2020</Year><Month>04</Month><Day>01</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2020</Year><Month>4</Month><Day>11</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2020</Year><Month>4</Month><Day>11</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2020</Year><Month>4</Month><Day>11</Day><Hour>6</Hour><Minute>1</Minute></PubMedPubDate></History><PublicationStatus>epublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">32272704</ArticleId><ArticleId IdType="pii">plants9040466</ArticleId><ArticleId IdType="doi">10.3390/plants9040466</ArticleId><ArticleId 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Michigan</li></orgName></list><tree><country name="États-Unis"><region name="Michigan"><name sortKey="Mccoy, Austin G" sort="Mccoy, Austin G" uniqKey="Mccoy A" first="Austin G" last="Mccoy">Austin G. Mccoy</name></region><name sortKey="Blomquist, Cheryl" sort="Blomquist, Cheryl" uniqKey="Blomquist C" first="Cheryl" last="Blomquist">Cheryl Blomquist</name><name sortKey="Chilvers, Martin I" sort="Chilvers, Martin I" uniqKey="Chilvers M" first="Martin I" last="Chilvers">Martin I. Chilvers</name><name sortKey="Martin, Frank N" sort="Martin, Frank N" uniqKey="Martin F" first="Frank N" last="Martin">Frank N. Martin</name><name sortKey="Miles, Timothy D" sort="Miles, Timothy D" uniqKey="Miles T" first="Timothy D" last="Miles">Timothy D. Miles</name><name sortKey="Woods, Patrick" sort="Woods, Patrick" uniqKey="Woods P" first="Patrick" last="Woods">Patrick Woods</name></country><country name="Canada"><noRegion><name sortKey="Bilodeau, Guillaume J" sort="Bilodeau, Guillaume J" uniqKey="Bilodeau G" first="Guillaume J" last="Bilodeau">Guillaume J. Bilodeau</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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HfdIndexSelect -h $EXPLOR_AREA/Data/Main/Exploration/RBID.i -Sk "pubmed:32272704" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd \
| NlmPubMed2Wicri -a PhytophthoraV1
| This area was generated with Dilib version V0.6.38. |  |