Development and Application of a Duplex RT-RPA Assay for the Simultaneous Detection of Cymbidium mosaic virus and...

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.