Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus.
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 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.
Dukes
J P
JP
Institute for Animal Health, Pirbright Laboratory, Pirbright, UK. Juliet.dukes@bbsrc.ac.uk
King
D P
DP
Alexandersen
S
S
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005453:Fluorescence; D005536:Foot-and-Mouth Disease / Q000175:diagnosis*; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000235:genetics
D000956:Antigens, Viral; D017931:DNA Primers; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C103557:virus-infection-associated antigen, Foot-and-mouth disease virus; D012321:DNA-Directed RNA Polymerases