Molecular beacons: A new approach to plant virus detection

Molecular beacons are single-stranded nucleic acid molecules with a stem-lo op conformation. The stem portion consists of complementary sequences at th e 5' and 3' terminals of the molecule, while the loop portion consists of p robe sequences that are complementary to the target sequences of choice. A fluorescent moiety is attached to one end, while a quenching moiety is atta ched to the opposite end. Reverse transcription-polymerase chain reactions are carried out with primers that amplify specific genome sequences of inte rest, yielding targets complementary to their respective molecular beacons for subsequent detection. Here, we have designed four molecular beacons spe cific to the RNA-dependent RNA polymerase and coat protein genes of two orc hid viruses, namely Cymbidium mosaic virus (CymMV) and Odontoglossum ringsp ot virus (ORSV). This technology is successfully applied to detect as littl e as 0.5 ng of viral RNA of both orchid viruses simultaneously in 100 mg of coinfected Oncidium orchid leaves. This rapid and specific technique is ap plicable to the orchid industry, which routinely carries out virus indexing and screening for virus-resistant cultivars. We belief that use of this mo lecular beacon approach can be extended to the detection of multiple plant viruses in various crops.