Development of colorimetric one-step reverse transcription loop-mediated isothermal amplification for rapid and visualized detection of tomato necrotic ringspot virus

Abstract

Tomato necrotic ringspot virus (TNRV) stands out as one of the most destructive viruses affecting chili and tomato crops in Thailand. TNRV causes typical necrotic and chlorotic ringspot symptoms on both leaves and fruits. This study focused on the development of a colorimetric one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid and visualized virus detection in different host plants, including chili, tomato, and physalis, a weed found near tomato fields. The new set of LAMP primers was designed based on the sequence of the non-structural (NSs) protein and nucleocapsid (N) protein genes of TNRV. The assay was optimized to identify the optimum isothermal temperature and incubation period. Positive reactions were identified by a color change from pink to yellow, while negative reactions remained pink where the assay worked well at 65°C for 30 min. Specificity and sensitivity assays were conducted. The results revealed that the optimal incubation time for detecting LAMP products, along with colorimetric changes, was 30 min at 65°C. The specificity assay demonstrated where LAMP primers did not cross-react with other orthotospoviruses like capsicum chlorosis virus (CaCV), melon yellow spot virus (MYSV) and watermelon silver mottle virus (WSMoV). The sensitivity assay indicated that RT-LAMP detected TNRV from RNA diluted up to 10–7 (1.0 fg/μL). Evaluation of the colorimetric one-step RT-LAMP assay on naturally infected tomato, chili, and physalis samples confirmed its successful detection of TNRV.