Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus
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Speed 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 °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.
KeywordsReverse Transcription Rapid Detection Diagnostic Sensitivity Single Tube Polymerase Gene
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- Anonymous (2002) The 2001 outbreak of foot and mouth disease. Report by the Controller and Auditor General. National Audit Office HC 939 Session 2001–2002: 21 June 2002Google Scholar
- Callahan, JD, Brown, F, Osorio, FA, Sur, JH, Kramer, E, Long, GW, Lubroth, J, Ellis, SJ, Shoulars, KS, Gaffney, KL, Rock, DL, Nelson, WM 2002Use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virusAm Vet Med Assoc22016361642CrossRefGoogle Scholar
- Coetzer, JAW, Thompson, GR, Tustin, RC, Kriek, NPJ 1994Foot-and-mouth diseaseCoetzer, JAWThomson, GRTustin, RCKriek, NPJ eds. Infectious diseases of livestock with special reference to Southern AfricaOxford University PressCape Town825852Google Scholar
- Ferris NP, King DP, Reid SM, Shaw AE, Hutchings GH (2006) Analysis of original laboratory results and retrospective analysis by real-time RT-PCR of virological samples collected from confirmed cases suggest over-reporting of foot-and-mouth disease in the UK in 2001. Vet Rec (in press)Google Scholar
- Iwasaki, MY, Yonekawa, T, Otsuka, K, Suzuki, W, Nagamine, K, Hase, T, Tatsumi, K-I, Horigome, T, Notomi, T, Kanda, H 2003Validation of the loop-mediated isothermal amplification method for single nucleotide polymorphism genotyping with whole bloodGenome Lett2119126Google Scholar
- King, DP, Ferris, NP, Shaw, AE, Reid, SM, Hutchings, GH, Giuffre, AC, Robida, JM, Callahan, JD, Nelson, WM, Beckham, TR 2005Detection of foot-and-mouth disease virus: comparative diagnostic sensitivity of two independent real-time RT-PCR assaysJ Vet Diagn Invest189296Google Scholar