Rapid , simple , field - deployable FMDV detection Juliet

Loop-mediated amplification (LAMP) amplifies specific nucleotide sequences but unlike PCR, it runs at a single temperature and positive reactions can be visualized with the naked eye so fragile instrumentation is not required. We have developed a one step, single-tube, accelerated, FMDVspecific RT-LAMP assay for the rapid detection of all seven serotypes of the virus, using primers designed against the 3D RNA polymerase-encoding region. RT-LAMP results were visualised by adding 5μl of the intercalating dye Picogreen. RT-LAMP was transferred to the Stratagene MX4000 real-time and analytical sensitivity was determined by using an in vitro transcribed standard and directly compared with a diagnostic TaqMan assay. Diagnostic sensitivity was assessed by testing samples of epithelia (n=98) submitted to the FAO WRL for FMD using both assays. From 10 copies of starting material, RT-LAMP product was detected in 15 minutes whilst 10 copies were detected in 22.2 minutes. Although TaqMan had similar analytical sensitivity, amplification took 50 minutes to achieve similar results. RT-LAMP detected FMDV in 81/98 of the field samples tested. Three virus isolates from suspect cases of FMD previously designated as negative by TaqMan were detected by RT-LAMP using specific primers. In contrast, thirteen samples designated positive by TaqMan, were not amplified by RT-LAMP. FMDV-specific RT-LAMP is carried out in a single tube, at 65°C for less than an hour. The real-time RT-LAMP assay for FMDV has a dynamic range over nine orders of magnitude. The assay can detect 10 copies of RNA template which is equivalent sensitivity to real-time RT-PCR. However, RTLAMP is faster than real-time RT-PCR and appears to be a rapid, sensitive, and cost-effective method for the detection of FMDV genomes at the pen-side, in the field and by developing countries.