Simultaneous quantitation of two orchid viruses by the TaqMan real-time RT-PCR

Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out using the TaqMan((R)) real-time RT-PCR, a novel detection technique that combines RT-PCR with the power of fluorescent detection. Four TaqMan((R)) probes were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of both viruses. The reporter dye FAM (6-carboxyfluorescein) was used to label the 5' terminus of probes specific to CymMV, while TET (tetrachloro-6-carboxyfluorescein) was used for the ORSV probes. TAMRA (6-carboxy-tetramethyl-rhodamine), which was attached at the 3' terminus of each probe, was used as the universal quencher. With increasing amounts of standard RNA templates, the respective threshold cycle (C(T)) values were determined and a linear relationship was established between these C(T) values and the logarithm of initial template amounts. The amounts of starting templates in mixed-infected Oncidium flowers and leaves were estimated from the standard curves. As little as 10(4) copies or 5 fg each of CymMV and ORSV could be detected simultaneously with either the RdRp or CP gene as the target. This system offers a sensitive, high throughput and rapid method for plant virus detection.