[Development of a rapid detection system for tomato spotted wilt orthotospovirus based on multienzyme isothermal...

Tomato spotted wilt virus (TSWV) is characterized by rapid transmission and a broad host range, creating an urgent need for detection methods applicable on-site at ports, in greenhouses, and in open fields. This study aims to develop a sensitive, specific, rapid, and field-applicable TSWV detection technique to provide technical support for the early diagnosis and precise monitoring of this virus. It targeted the conserved region of the TSWV L gene and designed multiple MIRA primers and probes. Through orthogonal-reciprocal cross screening, the optimal primer-probe combination was identified, and a 25 μL multienzyme isothermal rapid amplification (MIRA) assay was established. The assay detected viral RNA at a concentration as low as 1.984× 10^-4 ng/μL within 20 min at 37 °C. Its sensitivity was approximately 100-fold higher than that of conventional RT-PCR, and no cross-reactivity was observed with five related viruses, including ToCV and TYLCV , demonstrating high specificity. The MIRA-based method is simple to operate, rapid, and does not require large-scale precision instruments, enabling on-site detection. It provides a replicable technical paradigm for low-cost field surveillance and precise prevention and control of TSWV.