Development of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzae

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cg.authorship.typesCGIAR and developing country instituteen
cg.authorship.typesCGIAR and advanced research instituteen
cg.contributor.affiliationInternational Centre of Insect Physiology and Ecologyen
cg.contributor.affiliationUniversity of Nairobien
cg.contributor.affiliationJulius Kühn Instituten
cg.contributor.affiliationKenyatta Universityen
cg.contributor.affiliationWashington State Universityen
cg.contributor.affiliationInternational Livestock Research Instituteen
cg.contributor.affiliationUniversity of Bernen
cg.contributor.crpLivestock
cg.coverage.regionEastern Africa
cg.creator.identifierChris Jones: 0000-0001-9096-9728
cg.creator.identifierLillian Wambua: 0000-0003-3632-7411
cg.creator.identifierDaniel Masiga: 0000-0001-7513-0887
cg.creator.identifierJoerg Jores: 0000-0003-3790-5746
cg.creator.identifierCassandra Olds: 0000-0001-5244-2001
cg.howPublishedFormally Publisheden
cg.identifier.doihttps://doi.org/10.1016/j.mcp.2017.06.004en
cg.isijournalISI Journalen
cg.issn0890-8508en
cg.journalMolecular and Cellular Probesen
cg.reviewStatusPeer Reviewen
cg.subject.ilriCROPSen
cg.subject.ilriFODDERen
cg.subject.ilriFORAGESen
cg.volume35en
dc.contributor.authorWambua, Lillianen
dc.contributor.authorSchneider, B.en
dc.contributor.authorOkwaro, A.en
dc.contributor.authorWanga, J.O.en
dc.contributor.authorImali, O.en
dc.contributor.authorWambua, P.N.en
dc.contributor.authorAgutu, L.en
dc.contributor.authorOlds, Cassandra L.en
dc.contributor.authorJones, Christopher S.en
dc.contributor.authorMasiga, Daniel K.en
dc.contributor.authorMidega, C.A.O.en
dc.contributor.authorKhana, Z.en
dc.contributor.authorJores, Joergen
dc.contributor.authorFischer, A.en
dc.date.accessioned2017-07-05T14:54:25Zen
dc.date.available2017-07-05T14:54:25Zen
dc.identifier.urihttps://hdl.handle.net/10568/82664
dc.titleDevelopment of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzaeen
dcterms.abstractNapier grass Stunt Disease (NSD) is a severe disease of Napier grass (Pennisetum purpureum) in Eastern Africa, caused by the leafhopper-transmitted bacterium Candidatus Phytoplasma oryzae. The pathogen severely impairs the growth of Napier grass, the major fodder for dairy cattle in Eastern Africa. NSD is associated with biomass losses of up to 70% of infected plants. Diagnosis of NSD is done by nested PCR targeting the phytoplasma DNA, which is difficult to perform in developing countries with little infrastructure. We report the development of an easy to use, rapid, sensitive and specific molecular assay for field diagnosis of NSD. The procedure is based on recombinase polymerase amplification and targets the imp gene encoding a pathogen-specific immunodominant membrane protein. Therefore we followed a two-step process. First we developed an isothermal DNA amplification method for real time fluorescence application and then transferred this assay to a lateral flow format. The limit of detection for both procedures was estimated to be 10 organisms. We simplified the template preparation procedure by using freshly squeezed phloem sap from Napier grass. Additionally, we developed a laboratory serological assay with the potential to be converted to a lateral flow assay. Two murine monoclonal antibodies with high affinity and specificity to the immunodominant membrane protein IMP of Candidatus Phytoplasma oryzae were generated. Both antibodies specifically reacted with the denatured or native 17 kDa IMP protein. In dot blot experiments of extracts from infected plant, phytoplasmas were detected in as little as 12,5 μg of fresh plant material.en
dcterms.accessRightsOpen Access
dcterms.audienceScientistsen
dcterms.bibliographicCitationWambua, L., Schneider, B., Okwaro, A., Wanga, J.O., Imali, O., Wambua, P.N., Agutu, L., Olds, C., Jones, C.S., Masiga, D., Midega, C., Khan, Z., Jores, J. and Fischer, A. 2017. Development of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzae. Molecular and Cellular Probes 35: 44-56en
dcterms.extentp. 44-56en
dcterms.issued2017-10
dcterms.languageen
dcterms.licenseCC-BY-NC-ND-4.0
dcterms.publisherElsevieren
dcterms.subjectcropsen
dcterms.subjectforageen
dcterms.subjectnapier grass stunt diseaseen
dcterms.subjectpennisetum purpureumen
dcterms.subjectmycoplasmosesen
dcterms.typeJournal Article

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