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.

Development and Evaluation of a Fluorogenic 5′ Nuclease Assay To Detect and Differentiate between Ebola Virus Subtypes Zaire and Sudan

“…This assay is based on the 5Ј nuclease assay, which exploits the 5Ј nuclease activity of rTth polymerase to cleave a nonextendable, dual-labeled fluorogenic probe that is annealed to the target sequence during amplification (4,7,12). We chose the GP gene of Ebola virus as an assay target for several reasons.…”

Orchid Biology: Reviews and Perspectives, X

“…By this method, a quantity as low 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 (Eun, Seoh, and Wong, 2000). Taqman ® probes were synthesized, each targeting the RdRp and CP genes of CymMV and ORSV.…”

The Identification and Detection of Spongospora subterranea and Factors Affecting Infection and Disease

“…Real-time PCR is a technique in which DNA of a specific target organism can be quantified by measurements of the intensity of fluorescence with time during the exponential phase of DNA amplification. This method has been used with success for the detection and quantification of a range of plant pathogens including fungi (Bates et al 2001;Cullen et al 2001), bacteria (Hyman et al 2000) and viruses (Boonham et al 2000;Eun et al 2000). In this work a sensitive and quantitative realtime PCR assay specific to S. subterranea (van de Graaf et al 2003) was used as a tool for the detection and quantification of this important potato pathogen and virus vector in plant tissue, water and soil.…”