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.
We have developed a piezoelectric DNA-sensor based on DNA-RNA hybridization for the detection of two orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Specific oligonucleotide probes modified with a mercaptohexyl group at the 5'-phosphate end were directly immobilized onto 10-MHz AT-cut quartz crystal microbalance (QCM). QCMs coated with such oligonucleotide probes were exposed to test solutions containing viral RNA for hybridization. Various experimental conditions evaluated were (i) DNA probe coating concentration, (ii) sensitivity and specificity of the probes at different hybridization temperatures, and (iii) effects of incubation temperature on the hybridization time. The specific nucleotide probe-coated QCM-based DNA sensors were able to detect both CymMV and ORSV in quantities as low as approximately 1 ng in purified RNA preparations and 10 ng in the crude sap of infected orchids. This is the first application of a DNA biosensor for the detection of plant viruses.
Molecular beacons are single-stranded nucleic acid molecules with a stem-loop conformation. The stem portion consists of complementary sequences at the 5' and 3' terminals of the molecule, while the loop portion consists of probe sequences that are complementary to the target sequences of choice. A fluorescent moiety is attached to one end, while a quenching moiety is attached to the opposite end. Reverse transcription-polymerase chain reactions are carried out with primers that amplify specific genome sequences of interest, yielding targets complementary to their respective molecular beacons for subsequent detection. Here, we have designed four molecular beacons specific to the RNA-dependent RNA polymerase and coat protein genes of two orchid viruses, namely Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). This technology is successfully applied to detect as little 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 applicable to the orchid industry, which routinely carries out virus indexing and screening for virus-resistant cultivars. We belief that use of this molecular beacon approach can be extended to the detection of multiple plant viruses in various crops.
Immuno-capillary zone electrophoresis (I-CZE) is a technique that combines the specificity afforded by serological assays with the sensitivity, rapidity, and automation in detection provided by capillary zone electrophoresis. Cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) were detected in their purified forms as well as in the crude saps of infected Nicotiana benthamiana leaves and Oncidium orchid flowers. The two orchid virus-antibody complexes were resolved via the combined actions of electrophoretic migration and electro-osmotic flow along a buffer-filled, uncoated fused-silica capillary. The I-CZE fractions collected from both CymMV- and ORSV-antibody complex peaks, as well as the RNA purified from them, retained their infectivity upon inoculation onto Chenopodium quinoa. I-CZE assays were able to detect as little as 10 fg each of both CymMV and ORSV in their purified forms as well as in the crude saps of infected N. benthamiana and Oncidium orchid. As multiple samples can be analyzed rapidly, I-CZE offers an ideal diagnostic technique for routine mass-indexing programs such as virus-free certification, breeding for virus-resistant cultivars, plant quarantine, and germ plasm screening. This is the first report of the application of I-CZE for the detection of plant viruses.
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