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Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus
journal contribution
posted on 2006-06-01, 00:00 authored by J P Dukes, D P King, Soren AlexandersenSoren AlexandersenSpeed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan assay, RT-LAMP appears to be sensitive, rapid, specific, and cost-effective, with the potential for field deployment and use by developing countries for FMDV surveillance.
History
Journal
Archives of virologyVolume
151Issue
6Pagination
1093 - 1106Publisher
SpringerLocation
Berlin, GermanyPublisher DOI
ISSN
0304-8608Language
engPublication classification
C1.1 Refereed article in a scholarly journalCopyright notice
2006, Springer-VerlagUsage metrics
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No categories selectedKeywords
AnimalsAntigens, ViralDNA PrimersDNA-Directed RNA PolymerasesElectrophoresis, Agar GelFluorescenceFoot-and-Mouth DiseaseFoot-and-Mouth Disease VirusNucleic Acid Amplification TechniquesRNA, ViralReverse Transcriptase Polymerase Chain ReactionSensitivity and SpecificityTemperatureTime FactorsViral Nonstructural ProteinsScience & TechnologyLife Sciences & BiomedicineVirologyPOLYMERASE CHAIN-REACTIONTIME PCR MACHINEREAL-TIMEREACTION ASSAYDIAGNOSIS
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