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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #324891

Research Project: NOVEL DISEASE CONTROL STRATEGIES FOR CELLULAR AND SUB-CELLULAR PATHOGENS

Location: Molecular Plant Pathology Laboratory

Title: Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

Author
item Hammond, Rosemarie
item Zhang, Shulu - Agdia

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/26/2016
Publication Date: 7/15/2016
Citation: Hammond, R., Zhang, S. 2016. Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification. Journal of Virological Methods. 236:62-67.

Interpretive Summary: Tomato chlorotic dwarf viroid (TCDVd), a small nucleic acid molecule, is a serious threat to tomato production worldwide as infection leads to reduced plant vigor, small and deformed fruit, and yield loss. TCDVd 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, including plants and seeds, but current methods for detection require costly equipment and specific training. For this reason, we developed a rapid, specific, sensitive, and easy to perform test for TCDVd infection in leaf and seed 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: A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP® Acceler8TM RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP® Acceler8TM assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection.