Establishment and evaluation of two MIRA rapid detection methods for tomato chlorosis virus (ToCV)

Tomato chlorosis virus (ToCV) represents a major threat to the tomato industry in China. Conventional Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays require expensive thermal cyclers and are poorly suited to on-site screening in fields or at ports of entry. Here, two multi-enzyme isothermal rapid amplification (MIRA) systems targeting the highly conserved minor capsid protein (CPm) region of ToCV were developed: (i) a basic MIRA-electrophoresis assay (B-MIRA), and (ii) a fluorescent MIRA-real-time assay (F-MIRA). Primers/probes, reaction temperature, sensitivity and specificity were systematically optimized, and performance was validated with field samples.

The basic assay completed amplification at 41 °C within 30 min, generating a single 250 bp band; the limit of detection (LOD) was 2.78 × 10^-1 pg/μL. The fluorescent assay reached its endpoint in 20 min at 37 °C using the optimal primer/probe set F2/R1 + P (Ct = 12.4 ± 0.3 min); LOD was 2.78 × 10^-1 pg/μL.

The B-MIRA and F-MIRA assays developed in this study can reliably detect ToCV within 20 min with high sensitivity. Free of bulky instrumentation or professional laboratories, the protocols can be directly applied to seedling quarantine, field-side early inspection and border control, offering an accurate and ready-to-use diagnostic tool for the green management of ToCV. © 2026 Society of Chemical Industry.