Development of a Rapid Recombinase Polymerase Amplification Assay for Detection of Cymbidium ringspot virus

Cymbidium ringspot virus (CymRSV) can pose a significant threat to both the orchid and legume industries due to its wide host range and diverse modes of transmission. Rapid, sensitive, accurate, and easy-to-use detection of CymRSV is crucial for controlling its spread. This study developed a recombinase polymerase amplification (RPA) assay for the specific detection of CymRSV under isothermal conditions. The assay operates at a constant temperature of 39°C for 30 min without the need for thermal cycling. The method demonstrated high specificity, with no cross-reactivity to other viruses affecting orchids or related families. Sensitivity testing revealed a detection limit of 10² copies or 0.3 fg, indicating that the designed specific primers were highly sensitive to virus detection. This RPA-based detection technique offers a reliable solution for rapid diagnostics in quarantine settings, as well as in laboratories and field environments where advanced precision instruments may not be available.