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
cg.contributor.affiliationInternational Livestock Research Instituteen
cg.contributor.donorCGIAR Trust Funden
cg.contributor.initiativeGenebanks
cg.coverage.countryEthiopia
cg.coverage.iso3166-alpha2ET
cg.coverage.regionAfrica
cg.coverage.regionEastern Africa
cg.creator.identifierChris Jones: 0000-0001-9096-9728en
cg.creator.identifierAlemayehu Teressa Negawo: 0000-0001-8974-5321en
cg.creator.identifierAlice Muchugi: 0000-0002-8901-1376en
cg.howPublishedFormally Publisheden
cg.identifier.doihttps://doi.org/10.35248/2157-7471.23.14.690en
cg.issn2157-7471en
cg.journalJournal of Plant Pathology and Microbiologyen
cg.reviewStatusPeer Reviewen
cg.subject.actionAreaGenetic Innovation
cg.subject.ilriAGRICULTUREen
cg.subject.ilriANIMAL FEEDINGen
cg.subject.ilriCROP-LIVESTOCKen
cg.subject.ilriFEEDSen
cg.subject.ilriFODDERen
cg.subject.ilriFORAGESen
cg.subject.impactAreaEnvironmental health and biodiversity
dc.contributor.authorDawit, Woubiten
dc.contributor.authorMulatu, Fikerteen
dc.contributor.authorEshete, Yesufen
dc.contributor.authorNegawo, Alemayehu T.en
dc.contributor.authorMuchugi, Aliceen
dc.contributor.authorJones, Christopher S.en
dc.date.accessioned2023-12-24T14:49:02Zen
dc.date.available2023-12-24T14:49:02Zen
dc.identifier.urihttps://hdl.handle.net/10568/135890
dc.titleReverse Transcriptase Loop-Mediated Isothermal Amplification Assays (RTLAMP) for the Detection of Alfalfa Mosaic Virus from Forage Cropsen
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
dcterms.accessRightsOpen Access
dcterms.audienceCGIARen
dcterms.audienceDonorsen
dcterms.audienceScientistsen
dcterms.available2023-09-29en
dcterms.bibliographicCitationDawit, W., Mulatu, F., Eshete, Y, Negawo, A.T., 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
dcterms.issued2023-09-29en
dcterms.languageen
dcterms.licenseCC-BY-4.0
dcterms.subjectforageen
dcterms.subjectanimal feedingen
dcterms.subjectlivestocken
dcterms.typeJournal Article

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