Rapid and sensitive detection of Senecavirus valles using multiplex colorimetric RT-LAMP assays: Development and evaluation

Seneca Valley virus (SVV), a member of the species Senecavirus valles , has a significant impact on the swine industry by causing vesicular lesions that are clinically indistinguishable from those of other vesicular diseases, particularly foot-and-mouth disease (FMD). Due to this similarity, outbreaks involving SVV often result in the suspension of animal movements until FMD can be ruled out, as well as in temporary bans on pork exports. Rapid molecular diagnostic methods, such as RT-LAMP (reverse transcription loop-mediated isothermal amplification), are essential to minimise economic losses associated with suspected FMD outbreaks. In this study, three RT-LAMP primer sets were evaluated, targeting the VP1, VP2, and 3D regions of the SVV genome. While the 3D and VP2 primer sets were adapted from previously published primers, the VP1 set was newly designed. Optimal amplification was achieved using the dual-target VP1 + 3D primer combination after 40 minutes of incubation at 65 °C in the colorimetric phenol red assay. The integration of a 3D-printed RNA extraction cassette with a colorimetric RT-LAMP assay provides a streamlined point-of-care diagnostic workflow requiring only minimal equipment. Specificity analyses further demonstrated its potential as a rapid and low-cost alternative to RT-qPCR for SVV diagnosis.