Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection of foot-and-mouth disease virus

This paper describes the evaluation of four novel real-time multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for rapid and sensitive diagnosis of foot-and-mouth disease (FMD). In order to overcome the genetic diversity of FMD viruses (FMDV), these multiplex RT-LAMP assay pairs were established by combining four newly designed primer sets with two primer sets that had been previously published. Using a real-time turbidimeter to detect amplification products and a panel of 300 samples collected throughout the world over a 78-year period, the performance of the multiplex RTLAMP assays was compared with a FMDV-specific real-time RT-PCR assay. The most successful of the four multiplex RT-LAMP assays achieved a diagnostic sensitivity and specificity of 98.0% and 98.1%, and did not falsely detect FMDV in known negatives or samples containing swine vesicular disease virus, vesicular stomatitis virus or vesicular exanthema of swine virus. Furthermore, the analytical sensitivity of this multiplex RT-LAMP assay was at least as good as the individual component RT-LAMP tests. This is the first report of the development of a multiplex RT-LAMP to accommodate the high sequence variability encountered in RNA virus genomes and these results support the use of RT-LAMP as a cost-effective tool for simple diagnosis of FMD.