A portable recombinase polymerase amplification assay for the rapid detection of cucurbit leaf crumple virus in watermelon leaves and fruits

Autor: Pamela D. Roberts, Robert C. Hochmuth, Josh Freeman, Mathews L. Paret, Katherine Hendricks, Melanie Kalischuk
Rok vydání: 2021
Předmět:
Zdroj: Journal of Plant Pathology. 104:215-224
ISSN: 2239-7264
1125-4653
Popis: Cucurbit leaf crumple virus (CuLCrV), a single-stranded DNA virus belonging to the species Cucurbit leaf crumple virus in the genus Begomovirus, causes cucurbit leaf crumple disease in many species of the Cucurbitaceae family. This report describes the development and standardization of field-based isothermal recombinase polymerase amplification (RPA) and exonuclease RPA (exo-RPA) assays for the detection and diagnosis of CuLCrV in the leaves of watermelon, squash and pumpkin, and the fruits of watermelon. The oligonucleotide primers were made to target a portion of the BL1 movement protein. The primer set used in the RPA was highly specific for CuLCrV and did not show a cross-reaction with other cucurbit viruses nor in tissues with co-infections of CuLCrV with cucurbit yellow stunting disorder virus (CYSDV) and/or squash vein yellowing virus (SqVYV). These assays were sensitive, producing a positive signal with 1 fg/uL of purified total DNA and at a dilution of 1 × 10–5 of crude extract from CuLCrV-infected watermelon plants. Incorporation of a 6-fluorescein amidite (6-FAM) probe into the exo-RPA assay enabled a total sample processing and detection time of 30 min and positive reactions were detectable in different tissues including watermelon leaves, fruits, and pips (seeds lacking an embryo). The exo-RPA was efficiently used in the detection of CuLCrV in 21 cucurbit plants growing in different regions of southeastern U.S. A sensitive, rapid and easy field-based method for the detection and diagnosis of CuLCrV in watermelon was developed.
Databáze: OpenAIRE
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