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

Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexviru s (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out u sing the TaqMan((R)) real-time RT-PCR, a novel detection technique that com bines RT-PCR with the power of fluorescent detection. Four TaqMan((R)) prob es were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) a nd coat protein (CP) genes of both viruses. The reporter dye FAM (6-carboxy fluorescein) 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' term inus of each probe, was used as the universal quencher. With increasing amo unts 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 star ting 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 a s the target. This system offers a sensitive, high throughput and rapid met hod for plant virus detection. (C) 2000 Elsevier Science B.V. All rights re served.