Rapid identification of Tobacco rattle virus in potato and ornamental hosts using isothermal amplification and a hand-held fluorometric device

Tobacco rattle virus (TRV) infects hundreds of diverse plant species causing reduced quality and yield, particularly in potato and ornamental crops. Current detection methods are limited to commercial protein-based assays that do not target all infective isolates or published nucleic acid based assays that require significant training and equipment resources. Agdia Incorporated has commercialized isothermal recombinase polymerase amplification (RPA) technology that can be used for rapid and simple detection of plant pathogens. Innovations are needed to improve quality and lower cost in order to bring this technology to growers and laboratories with limited time and resources that also require early detection of this virus to enact disease management and phytosanitary decisions. Specific objectives of this project include: design of a fluorometric RPA assay that simultaneously reverse transcribes and amplifies both the target nucleic acid and an internal control, verification of the test sensitivity in the presence of multiple crude plant extracts and natural inhibitors, evaluation of component materials needed for a low-cost and reusable minifluorometric device, and verification of the device accuracy and precision in a range of field conditions. After addressing these challenges in Phase I, the TRV-specific test can be validated with regulatory and commercial stakeholders to evaluate robustness and determine if the test is fit for use in a broader market. Stability testing will determine whether the assay retains performance characteristics under variable and protracted shipping conditions to Agdia's international clients and distributors. Generation and evaluation of minifluorometer prototypes with manufacturing partner(s) will be needed for commercial release.