Trouble-free detection of grapevine leafroll-associated virus-3 employing reverse transcription-recombinase polymerase amplification assay

Grapevine leafroll-associated virus-3 (GLRaV-3) is a viral pathogen of grapevine leafroll disease (GLD) complex, responsible for debilitating viticulture across the globe. The only available options for management of this important virus are vector control and selection of clean plant propagating materials. To detect this virus in planting material and field samples, it is necessary to develop a quick, reliable, sensitive, and cost-effective detection method. In this study, a simple crude plant extract-based reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for efficient detection of GLRaV-3. This assay is quick and trouble-free in sample preparation and can be performed at a wide range of temperatures (20–45 °C) and time periods (20–35 min) although best results were obtained at 40 °C for 30 min of incubation. The assay does not require tedious RNA isolation as grapevine leaf sap simply prepared in NaOH: EDTA (1:1) buffer is directly used as template. The developed RT-RPA assay has been shown to be highly sensitive and specific for the detection of GLRaV-3 in grapevine samples, with a limit of detection as low as 10 –5 dilutions. Field validation and application of the developed assay further confirmed the sensitivity and reliability as 76% of samples were tested positive for GLRaV-3. The developed assay requires less than 60 min for its completion, making it a rapid and efficient tool for screening large numbers of grapevine samples and planting materials. The simplicity and efficiency of the developed RT-RPA assay make it a promising tool for detecting GLRaV-3 in grapevines, allowing for timely and accurate diagnosis and control of this economically significant virus.