Field-portable, rapid, and low-cost RT-LAMP assay for the detection of tomato chlorotic spot virus.

Abstract

Tomato chlorotic spot virus (TCSV) is a highly destructive, thrips-transmitted, emerging orthotospovirus in various vegetable and ornamental crops. It is important to reduce the risk of spreading this virus by limiting the movement of infected plant materials to other geographic areas by utilizing point-of-care diagnostics. Current diagnostic assays for TCSV require costly lab equipment, skilled personnel, and electricity. Here we report the development of a simple rechargeable battery-operated handwarmer-assisted reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay and demonstrate a step-by-step protocol to achieve in-field detection of TCSV. Under field conditions, handwarmer-assisted RT-LAMP can detect as little as 0.9 pg/μl of total RNA from TCSV-infected tomato plants in <35 minutes. When fully charged, the field-portable device can be used in six consecutive RT-LAMP detection assays, yielding test results for 96 individual samples. Dye-based colorimetric methods, including pH and metal ion indicators, were evaluated to eliminate laboratory-dependent LAMP visualization. Phenol-red combined with hydroxynaphthol blue was adopted in the handwarmer-assisted RT-LAMP detection method to obtain a more robust color difference distinguishable by the naked eye. Overall, handwarmer-assisted RT-LAMP is a rapid, highly sensitive, and cost-effective diagnostic technique that can be used by non-specialist personnel in the field, particularly in rural production areas lacking access to a diagnostic lab or constant electricity.