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

• Published in: Journal of virological methods 2007-12, Vol.146 (1), p.155-164
• Main Authors: Miglino, Roberto, Jodlowska, Agata, Pappu, Hanu R., van Schadewijk, Ton R.
• Format: Article
• Language: English
• 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
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2007.06.014

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.