Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are among the world’s most serious and widespread orchid viruses; they often infect orchids, causing devastating losses to the orchid industry. Therefore, it is critical to establish a method that can rapidly and accurately detect viruses in the field using simple instruments, which will largely reduce the further spread of viruses and improve the quality of the orchid industry and is suitable for mass promotion and application at grassroots agrotechnical service points. In this investigation, we established a rapid amplification method for virus detection at 39 °C for 35 min to detect the presence of CymMV and ORSV simultaneously, sensitively, and specifically in orchids. Primers for the capsid protein (CP)-encoding genes of both viruses were designed and screened, and the reaction conditions were optimized. The experimental amplification process was completed in just 35 min at 39 °C. There were no instances of nonspecific amplification observed when nine other viruses were present. The RPA approach had detection limits of 10⁴ and 10³ copies for pMD19T-CymMV and pMD19T-ORSV, respectively. Moreover, the duplex RT-RPA investigation confirmed sensitivity and accuracy via a comparison of detection results from 20 field samples with those of a gene chip. This study presents a precise and reliable detection method for CymMV and ORSV using RT-RPA. The results demonstrate the potential of this method for rapid virus detection. It is evident that this method could have practical applications in virus detection processes.

Cymbidium ringspot virus (CymRSV) can pose a significant threat to both the orchid and legume industries because of 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 10 ² 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.