A High-Fidelity RT-LAMP Molecular Diagnostic Assay for Rapid Detection of Sunflower Stem Necrosis Disease.

Abstract

Sunflower necrosis disease (SND), caused by Tobacco streak virus (TSV), spreads rapidly through infected pollen, mechanical sap transmission, and thrips vectors, leading to severe yield losses in sunflower and other crops. Hence, early and accurate detection of TSV is essential for effective surveillance and timely disease management. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and validated for the detection of TSV. Total RNA was extracted and subsequently reverse transcribed into complementary DNA (cDNA), which served as the template for amplification. The assay used six primers targeting the coat protein (CP) gene and reaction conditions were optimized for magnesium sulfate, betaine, dNTPs, temperature, and incubation time. Amplification was visualized by hydroxynaphthol blue (HNB) dye and confirmed through agarose gel electrophoresis. The assay specifically detected TSV without cross-reactivity to other RNA viruses. The assay demonstrated a detection limit of 0.05 pg of cDNA, comparable to conventional RT-PCR and was able to successfully detect TSV in both symptomatic and asymptomatic plants. These results highlight the assay as a rapid, sensitive, and reliable tool for early virus detection and effective disease management.