A reverse transcription-recombinase polymerase amplification (RT-RPA) assay for rapid and sensitive detection of tobacco streak virus causing sunflower necrosis disease.

Abstract

Sunflower necrosis disease (SND), caused by tobacco streak virus (TSV), is an economically important disease of sunflower. Rapid and sensitive detection of TSV is crucial for the management of SND. In the present study, we developed an isothermal amplification assay based on the reverse transcription-recombinase polymerase amplification (RT-RPA) technique for the first time for rapid and sensitive detection of TSV using crude leaf sap of infected plants, obtained by crushing the samples in Tris extraction buffer (pH 7.5, 1 M) as a template. For reliable detection of TSV in crude leaf sap using the developed assay, the reaction mixture was incubated at 38 °C for 30 min. The developed assay was specific to the TSV and detected the virus up to 10^-8, 10^-12, and 10^-13 dilutions of crude sap, cDNA, and plasmid template, respectively. The detection threshold limit of TSV by RPA assay was up to 1000 times more sensitive (cDNA and plasmid used as templates) than reverse transcription polymerase chain reaction (RT-PCR). Using the developed RT-RPA assay, TSV was detected in 23 field samples, including five asymptomatic samples of sunflower germplasm accessions grown at ICAR-NBPGR Regional Station, Hyderabad, during the kharif 2024, thereby demonstrating the suitability of the assay for rapid detection of TSV from field-collected samples.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04419-x.