Development of a real-time duplex isothermal assay for the detection of Tobacco rattle virus and an endogenous internal RNA control in ornamental hosts
Tobacco rattle virus (TRV) is capable of infecting upwards of 400 plant species including prominent ornamentals such as Dicentra, peony, Hosta, Epimedium, Clematis, Astilbe, Heuchera, tulip, and Petunia. The virus can often be present in these hosts yet remain symptomless. 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. We present a novel isothermal diagnostic tool for the detection of TRV. The isothermal assay utilizes recombinase polymerase amplification to detect over 25 diverse TRV isolates in a 20-min real-time detection scheme with comparable sensitivity to existing real-time and conventional PCR methods. The assay incorporates an endogenous RNA internal control to identify when inhibitors from fragrant ornamentals, succulents, and starchy potato tubers cause a false negative diagnosis 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 better detection of TRV across different hosts in diverse environments.
Davenport, B., Groth-Helms, D., Li, R., Zhang, S., Gray, S., Ingram, J. and Pappu, H. (2020). Development of a real-time duplex isothermal assay for the detection of Tobacco rattle virus and an endogenous internal RNA control in ornamental hosts. Acta Hortic. 1288, 245-255
ornamentals, recombinase polymerase amplification, TRV, virus detection