A rapid fluorescence-based real-time isothermal assay for the detection of Cucurbit yellow stunting disorder virus in squash and watermelon plants

• Published in: Molecular and cellular probes Vol. 53; p. 101613
• Main Authors: Kalischuk, Melanie L., Roberts, Pamela D., Paret, Mathews L.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.10.2020
• Subjects: Citrullus lanatus; Cucurbit yellow stunting disorder; Cucurbits; Isothermal amplification; Recombinase polymerase amplification; Cucurbit yellow stunting disorder; Cucurbits; Isothermal amplification; Citrullus lanatus; Recombinase polymerase amplification
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2020.101613

Cucurbit yellow stunting disorder virus (CYSDV) is a single-stranded positive-sense RNA virus that produces devastating disease in watermelon and squash. Foliar symptoms of CYSDV consist of interveinal yellowing, brittleness, and thickening of older leaves leading to reduced plant vigor. A rapid diagnostic method for CYSDV would facilitate early detection and implementation of best viral-based management practices. We developed a rapid isothermal reverse transcription-recombination polymerase amplification (exo RT-RPA) assay for the detection of CYSDV. The primers and a 6-fluorescein amidite (6-FAM) probe were developed to target the nucleocapsid gene. The real-time assay detected CYSDV at 2.5 pg purified total RNA extracted from CYSDV-infected leaf tissue and corresponded to 10 copies of the target molecule. The assay was specific and did not cross-react with other common cucurbit viruses found in Florida and Georgia. The performance of the exo RT-RPA was evaluated using crude extract from 21 cucurbit field samples and demonstrated that the exo RT-RPA is a rapid procedure, thus providing a promising novel alternative approach for the detection of CYSDV. •An exo RT-RPA assay developed for simple and rapid detection of Cucurbit yellow stunting disorder virus (CYSDV).•The exo RT-RPA primers/probe were highly specific and sensitive in the detection of CYSDV.•The assay detected CYSDV from crude extract of watermelon and squash leaves.