Reverse Transcriptase Loop-Mediated Isothermal Amplification Assays (RTLAMP) for the Detection of Alfalfa Mosaic Virus from Forage Crops

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cg.authorship.typesCGIAR single centreen_US
cg.contributor.affiliationInternational Livestock Research Instituteen_US
cg.contributor.donorCGIAR Trust Funden_US
cg.contributor.initiativeGenebanksen_US
cg.coverage.countryEthiopiaen_US
cg.coverage.iso3166-alpha2ETen_US
cg.coverage.regionAfricaen_US
cg.coverage.regionEastern Africaen_US
cg.creator.identifierChris Jones: 0000-0001-9096-9728en_US
cg.creator.identifierAlemayehu Teressa Negawo: 0000-0001-8974-5321en_US
cg.creator.identifierAlice Muchugi: 0000-0002-8901-1376en_US
cg.howPublishedFormally Publisheden_US
cg.identifier.doihttps://doi.org/10.35248/2157-7471.23.14.690en_US
cg.issn2157-7471en_US
cg.journalJournal of Plant Pathology and Microbiologyen_US
cg.reviewStatusPeer Reviewen_US
cg.subject.actionAreaGenetic Innovationen_US
cg.subject.ilriAGRICULTUREen_US
cg.subject.ilriANIMAL FEEDINGen_US
cg.subject.ilriCROP-LIVESTOCKen_US
cg.subject.ilriFEEDSen_US
cg.subject.ilriFODDERen_US
cg.subject.ilriFORAGESen_US
cg.subject.impactAreaEnvironmental health and biodiversityen_US
dc.contributor.authorDawit, Woubiten_US
dc.contributor.authorMulatu, Fikerteen_US
dc.contributor.authorEshete, Yesufen_US
dc.contributor.authorNegawo, Alemayehu T.en_US
dc.contributor.authorMuchugi, Aliceen_US
dc.contributor.authorJones, Christopher S.en_US
dc.date.accessioned2023-12-24T14:49:02Zen_US
dc.date.available2023-12-24T14:49:02Zen_US
dc.identifier.urihttps://hdl.handle.net/10568/135890en_US
dc.titleReverse Transcriptase Loop-Mediated Isothermal Amplification Assays (RTLAMP) for the Detection of Alfalfa Mosaic Virus from Forage Cropsen_US
dcterms.abstractAMV is one of the most economically important plant viruses and has a very wide host range including forage crops. To make sure that only seeds of satisfactory phytosanitary status are distributed to recipients in geographically diverse areas, the ILRI genebank routinely monitors seed-borne diseases during seed multiplication and in the field genebank. Current detection techniques for AMV include a dot-blot assay and a two-step Reverse Transcription Polymerase Chain Reaction (RT-PCR), both of which are time consuming. In the present study we developed a one step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) method for the sensitive and rapid detection of AMV from forage crops. For the assay, six different AMV specific primers were used, and a series of reactions were performed to identify optimal conditions. Amplicons were easily visualized by means of an in-tube colour indicator (SYBR Green I dye) with no requirement to run gel electrophoresis. The sensitivity of the RT-LAMP assay was assessed by comparing the optimized AMV RT-LAMP assay with the conventional RT-PCR. In RT-LAMP, an amplicon was generated up to 100 ag/μl dilutions in contrast to the conventional RT-PCR, where no amplification at 1 fg/μl and onwards, indicating that the detection limit of the AMV RT-LAMP assay is much lower than for the conventional RT-PCR. Finally, the optimized RT-LAMP assay was further validated on 40 field samples of different forage species and other important forage viruses. The developed RT-LAMP assay was specific and detected AMV from different forages with no cross reaction with other plant viruses (SBMV and potyvirus) tested in the ILRI-genebank seed multiplication/regeneration fields. The optimized RT-LAMP assay is an effective tool for the detection of AMV for field samples in diagnostic laboratories, and for quarantine applications.en_US
dcterms.accessRightsOpen Accessen_US
dcterms.audienceCGIARen_US
dcterms.audienceDonorsen_US
dcterms.audienceScientistsen_US
dcterms.available2023-09-29en_US
dcterms.bibliographicCitationDawit, W., Mulatu, F., Eshete, Y, Terresa, A., Muchugi, A. and Jones, C.S. 2023. Reverse Transcriptase Loop-Mediated Isothermal Amplification Assays (RTLAMP) for the Detection of Alfalfa Mosaic Virus from Forage Crops. Journal of Plant Pathology and Microbiology 14(5):1000690.en_US
dcterms.issued2023-09-29en_US
dcterms.languageenen_US
dcterms.licenseCC-BY-4.0en_US
dcterms.subjectforageen_US
dcterms.subjectanimal feedingen_US
dcterms.subjectlivestocken_US
dcterms.typeJournal Articleen_US

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ILRI feed and forage development program outputs
CGIAR Initiative on Genebanks
ILRI forage genebank:Team-wide outputs