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

Abstract

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.

Fig. 1

Genome organisation of (A) CymMV and (B) ORSV showing the RNA-dependent RNA polymerase (RdRp); triple gene block (TGB) 1, 2 and 3; movement protein (MP) and coat protein (CP). The positions of the four primer pairs (CymRdRp-F and CymRdRp-R, CymCP-F and CymCP-R, ORSRdRp-F and ORSRdRp-R, ORSCP-F and ORSCP-R) and the four TaqMan probes (CymRdRp, Cym-CP, ORS-RdRp, ORS-CP) are indicated.

Fig. 2

Quantification curves of (A) the RNA-dependent RNA polymerase gene and (B) coat protein gene of CymMV and ORSV. In vitro transcribed viral RNAs were serially diluted from 10⁹ to 10⁴ copies at one log unit intervals. The average threshold cycle (C_T) was plotted against the logarithm of the initial copy numbers (1.00E+n≡1×10ⁿ).

Fig. 3

The average initial number copy number of CymMV and ORSV RNA-dependent RNA polymerase and coat protein genes per ng of total nucleic acid extracted from Oncidium flowers or leaves. Standard deviations for each vertical bar were calculated from an average of ten replicates (1.00E+n≡1×10ⁿ).