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ISHS Acta Horticulturae 1242: III International Symposium on Horticulture in Europe - SHE2016

DiRT-qPCR for molecular biological detection of RNA encoded viruses

Authors:   J. Stammler, J. Hadersdorfer, A. Kellermann, D. Treutter
Keywords:   Potato leaf roll virus, TaqMan real-time PCR assay, simple sample preparation
DOI:   10.17660/ActaHortic.2019.1242.120
Abstract:
For propagation of certified horticultural but also agricultural plant material, a phytosanitary virus screening is obligatory in order to avoid the spread of plant viruses regionally but also globally. To date, most high throughput standard virus screenings are still based on the immunological detection method ELISA because of its robustness, high throughput potential and very good cost-benefit ratio. However, in some cases molecular biological applications are compulsorily. For instance in the process of seed potato certification, low virus titres present in seed potatoes require a long virus enrichment phase for adequate virus detection by DAS-ELISA. This time consuming step can be avoided by a change to more sensitive molecular biological techniques. Most nucleic acid based detection protocols are accompanied by high costs for their performance. We introduce a TaqMan qPCR based one step protocol, named direct reverse transcriptase quantitative PCR (DiRT-qPCR), for the detection of RNA encoded viruses without sophisticated nucleic acid purification at lower costs but with high throughput potential exemplarily on (+)ssRNA Potato leafroll virus (PLRV).

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