A semi-automated and highly sensitive streptavidin magnetic capture-hybridization RT-PCR assay: Application to genus-wide or species-specific detection of several viruses of ornamental bulb crops

A semi-automated and highly sensitive streptavidin magnetic capture-hybridization RT-PCR assay: Application to genus-wide or species-specific detection of several viruses of ornamental bulb crops

A semi-automated, rapid and sensitive method that combines magnetic capture-hybridization and reverse-transcription polymerase chain reaction (MCH/RT-PCR) for the detection of plant viruses is described. The assay uses a target specific biotin-labelled oligoprobe for RNA capture and streptavidin-coa...

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Bibliographic Details
Published in:Journal of virological methods Vol. 146; no. 1; pp. 155 - 164
Main Authors: Miglino, Roberto, Jodlowska, Agata, Pappu, Hanu R., van Schadewijk, Ton R.
Format: Journal Article
Language:English
Published: London Elsevier B.V 01-12-2007
Amsterdam Elsevier
New York, NY
Subjects:
Biological and medical sciences
Biotin - metabolism
Fundamental and applied biological sciences. Psychology
KingFisher automatic nucleic acid isolation station
Microbiology
Plant Diseases - virology
Plant Viruses - isolation & purification
Potexvirus
Reverse Transcriptase Polymerase Chain Reaction - methods
Sensitivity and Specificity
Streptavidin - metabolism
Techniques used in virology
Tobacco rattle virus
Tobravirus
Virology
Tobravirus
Potexvirus
KingFisher automatic nucleic acid isolation station
Microbiology
Species specificity
Method
Virology
Virus
Flexiviridae
Isolation
Detection
Reverse transcription polymerase chain reaction
Nucleic acid
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Summary:A semi-automated, rapid and sensitive method that combines magnetic capture-hybridization and reverse-transcription polymerase chain reaction (MCH/RT-PCR) for the detection of plant viruses is described. The assay uses a target specific biotin-labelled oligoprobe for RNA capture and streptavidin-coated magnetic beads for subsequent RNA-oligoprobe hybrid isolation from plant lysate. Detection and specific identification was accomplished by RT-PCR. This approach was investigated for the specific detection of Tobacco rattle virus and for the detection of viruses within the Potexvirus group in leaves, dormant bulbs and corms of flower bulbs of different species. Dilution series of TRV-infected tulip leaf sap showed that MCH/RT-PCR was 70,588 times more sensitive than enzyme-linked immunosorbent assay (ELISA) and was similar to that of RT-PCR. ELISA underestimated the infection levels of TRV in field samples compared to MCH/RT-PCR. The ability of MCH/RT-PCR to be performed in a microtiter plate on an automatic nucleic acid isolation station facilitates high throughput virus diagnostics. RNA isolation and purification was rapid, specific, sensitive, contamination-free and reproducible making this method amenable for routine indexing of stock plants as part of a management plan to reduce the propagation of virus-infected plants.
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ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2007.06.014