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.

葫芦科黄矮化病病毒（CYSDV）是一种单链正义RNA病毒，可在西瓜和南瓜中造成毁灭性疾病。CYSDV的叶面症状包括静脉变黄，变脆和较老的叶子变厚，导致植物活力降低。CYSDV的快速诊断方法将有助于及早发现和实施基于病毒的最佳管理方法。我们开发了一种快速等温逆转录重组聚合酶扩增（exo RT-RPA）测定法来检测CYSDV。开发了引物和6-荧光素酰胺（6-FAM）探针以靶向核衣壳基因。实时分析检测到从CYSDV感染的叶片组织中提取的2.5 pg纯化总RNA中的CYSDV，并对应于目标分子的10个拷贝。该测定是特异性的，不会与在佛罗里达州和乔治亚州发现的其他常见葫芦科病毒发生交叉反应。使用来自21个葫芦田野样品的粗提物评估exo RT-RPA的性能，并证明exo RT-RPA是一种快速程序，因此为检测CYSDV提供了一种有希望的新方法。