Development of a sensitive diagnostic assay to detect Cymbidium mosaic virus and Odontoglossum ringspot virus in members of the Orchidaceae

The Orchidaceae is a family of flowering plants native to China, members of which possess high ornamental and pharmacological value. Many members of the family Orchidaceae are susceptible to infection by both Odontoglossum ringspot tobamovirus (ORSV) and Cymbidium mosaic potexvirus (CymMV). Symptoms of viral infection include ringspots, mosaics, mottles, chlorotic streaks, and stripes on the leaves, and viral infections cause significant losses in the commercial value of these plants. However, there are no effective measures to control ORSV and CymMV. Therefore, it is vital to identify viral epidemics through the rapid and efficient detection of these two viruses, in order to limit their economic damage. We have developed an assay for detecting co-infection of members of the Orchidaceae by ORSV and CymMV. The coat protein (CP) genes from ORSV and CymMV were cloned and the CPs were expressed in vitro and used to generate two virus-specific polyclonal antisera that were validated by western blotting. Polyclonal anti-ORSV-CP and anti-CymMV-CP antisera were used simultaneously to develop a bi-immunocapture reverse transcription-polymerase chain reaction (BIC RT-PCR) assay. We demonstrated that the BIC RT-PCR assay was sensitive and specific, and provided a valuable tool for the early detection of ORSV and CymMV in leaf sap extracts from plants of the Orchidaceae