Tobacco rattle virus (TRV), the causal agent of corky ringspot in potato, is capable of infecting upwards of 400 plant species. Reliable detection of all infectious TRV isolates is often limited to molecular methods due to the expansive host range, genomic arrangement, and absence of the RNA2 segment in some isolates. Agdia presents a novel isothermal diagnostic tool for the detection of Tobacco rattle virus. The isothermal assay utilizes recombinase polymerase amplification to detect over 25 diverse TRV isolates in a 20-minute real-time detection scheme and matches the sensitivity of existing real-time and conventional PCR methods. The assay incorporates an endogenous RNA control to assess if inhibition from fragrant ornamentals, succulents, and starchy potato tubers occurs in molecular diagnostics of TRV. The assay demonstrates competency of target detection in over 70 plant species utilizing a crude extraction comparable to common serological assays. Paired with a portable fluorometer the assay will allow for a broader screen of TRV across different hosts in diverse environments.