Simultaneous TD/RT-PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers

Seoh, Mui-Leng; Wong, Sek‐Man; Zhang, Lee

doi:10.1016/s0166-0934(98)00018-4

Cited by 59 publications

(34 citation statements)

References 10 publications

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“…Furthermore, the efficiency of multiplex detection is significantly affected by the extension time and the concentrations of dNTPs, primers and MgCl 2 (Seoh et al 1998, James 1999. Vastly different levels of target mRNAs will cause quantification problems even for real-time detection, as the exponential phase of amplification of the less abundant mRNA will not overlap with that of the highly abundant target.…”

Section: Multiplex Rt-pcrmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,429

2,448

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The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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Section: Multiplex Rt-pcrmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,429

2,448

View full text Add to dashboard Cite

show abstract

“…tion methods based on targeting viral nucleic acids [such as reverse transcription-polymerase chain reaction (RT-PCR), hybridization and molecular beacon] (Lim et al 1993;Hu and Wong 1998;Seoh et al 1998;Lee and Chang 2006;Sherpa et al 2006), viral capsid proteins (CP) [such as enzyme-linked immunosorbent assay (ELISA), quartz crystal microbalance (QCM) immunosensors, immuno-capillary zone electrophoresis (I-CZE), liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption-ionization (MALDI) mass spectrometry] (Arunasalam and Pearson 1989;Wong and Chng 1993;Eun and Wong 1999;Tan et al 2000;Eun et al 2002) or both [such as immunocapture-PCR (IC-PCR)] (Barry et al 1996) were developed. Among these detection methods, ELISA is cost-effective and shows satisfying detection sensitivity (1-10 ng ml −1 ; Hull 2002).…”

mentioning

confidence: 99%

Performances and application of antisera produced by recombinant capsid proteins of Cymbidium mosaic virus and Odontoglossum ringspot virus

Lee

Chang

2008

Eur J Plant Pathol

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Antisera against important orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), were separately produced using bacterially expressed recombinant capsid proteins (CP), instead of purified virus particles, as immunogens. These antisera were then designated as home-made CymMV CP antiserum (HM-Cy) and home-made ORSV CP antiserum (HM-OR). The high specificity of HM-Cy and HM-OR were confirmed by immunoblot. Their detection limits were determined using indirect-enzyme-linked immunosorbent assay (I-ELISA). Both HM-Cy and HM-OR showed low background reactivity to healthy plants and thus displayed a high S/H ratio (sample OD405/healthy control OD405) in tested orchids. The data indicated that our antisera were efficient and accurate in determination of negative and positive results in ELISA test as commercial antibodies. Therefore, these homemade antisera of CymMV and ORSV are suitable for the certification programme of orchids due to their low cost and high specificity. HM-Cy and HM-OR were further used for a field survey to study the incidence of CymMV and ORSV. The results showed that CymMV is more prevalent than ORSV in Taiwan.

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“…Since many cultivated orchids tended to be infected by different viruses, such as the CyMV and ORSV [46]), the abundant virus transcripts in the plant cells and tissues might interfere with the SSH or the cDNA-AFLP analysis [47]. In this study, although limited numbers of differentially expressed ESTs were obtained, several interesting clones derived from both suppression subtractive hybridization and cDNA-RAPD analyses revealed potential roles in flower development.…”

Section: Discussionmentioning

confidence: 91%

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture

Chen

Tsai

et al. 2005

Cell Res

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Simultaneous TD/RT-PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers

Cited by 59 publications

References 10 publications

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Performances and application of antisera produced by recombinant capsid proteins of Cymbidium mosaic virus and Odontoglossum ringspot virus

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture

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