Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification

Authors: KOVALSKAYA, NATALIA - Orise Fellow; Hammond, Rosemarie

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/6/2021
Publication Date: 11/10/2021
Publication URL: https://handle.nal.usda.gov/10113/7564532
Citation: Kovalskaya, N., Hammond, R. 2021. Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification. Journal of Virological Methods. 300:114353. https://doi.org/10.1016/j.jviromet.2021.114353.
DOI: https://doi.org/10.1016/j.jviromet.2021.114353

Interpretive Summary: Tomato apical stunt viroid (TASVd), a small nucleic acid plant pathogen, is a serious threat to tomato production worldwide as infection leads to reduced plant vigor, small and deformed fruit, and yield loss. TASVd is seed-transmitted and is easily transmitted mechanically from plant to plant once introduced. Seed treatments do not control transmission of the disease. The most effective control includes the use of certified viroid-free planting materials, but current methods for detection require costly equipment and specific training. For this reason, we developed rapid, specific, sensitive, and easy to perform tests for TASVd infection in leaf tissues that can be used under field and laboratory conditions. This information will be of use to scientists, growers, the industry, and regulatory agencies, who are developing methods and protocols to control viroid diseases.

Technical Abstract: Tomato apical stunt viroid (TASVd) is a serious threat to tomato plants that can cause a considerable yield loss. In the present study, two isothermal molecular diagnostic assays based on reverse transcription-recombinase polymerase amplification (RT-RPA) utilizing the AmplifyRP® platform for plant pathogen detection were developed. The results of this research demonstrated distinct specificity of both developed assays, AmplifyRP® Acceler8™ and AmplifyRP® XRT, expressed in the absence of any cross-reaction activity to all total RNA extracts obtained from plants infected with other pospiviroids. The RT-RPA assays detected viroid RNA in 81- and 27-fold dilutions of the original TASVd-infected crude extract for AmplifyRP® Acceler8™ and AmplifyRP® XRT, respectively. The sensitivity tests in serial water dilutions showed the ability of AmplifyRP® Acceler8™ and AmplifyRP® XRT to detect 8 and 80 fg of pure TASVd RNA transcript, respectively. The influence of crude extract on viroid RNA transcript detection was also examined and a decrease of sensitivity of approximately 100-fold for both RT-RPA assays was revealed. To our knowledge, this is the first report describing development of RT-RPA assays to detect TASVd in plants using the AmplifyRP® platform that can be further employed both in laboratory conditions and in the field for on-site diagnosis.