1. Optimization of an isothermal recombinase polymerase amplification method for real-time detection of Potato virus Y O and N types in potato.
- Author
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Babujee, Lavanya, Witherell, Robert A., Mikami, Kakeru, Aiuchi, Daigo, Charkowski, Amy O., and Rakotondrafara, Aurélie M.
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POTATO virus Y , *FLUORESCENT probes , *MESSENGER RNA , *POTATOES , *SEED crops , *VIRAL genomes - Abstract
Highlights • Primers targeting the highly conserved pipo region specifically recognized different Potato virus Y isolates by RPA. • The RPA assay efficiently detects viral RNA in low viral titer samples, including the peridermal tissues of potato tubers. • The RPA procedure was streamlined by using crude extract from infected-potato tissues. • The real-time detection of Potato virus Y using fluorescent probe picked up the viral RNA within 6 min. Abstract Potato virus Y (PVY) is a global challenge for potato production and the leading cause of seed crop downgrading and rejection for certification. Accurate and timely diagnosis is key to effective control of PVY. Here we optimized the isothermal recombinase polymerase amplification (RPA) for accurate detection of different PVY O and N types that were tested, present in different tissues of potato plants including tubers with a primer set that specifically targets the highly conserved pipo region within the viral genome. Combined with a simplified preparation of the template by tissue homogenization, we established a rapid RPA procedure, which can allow real time detection in less than 10 min with a fluorescent probe. Specificity of the reaction was determined by the lack of cross-reactivity with other common potato viruses. Although RPA reagents remain more expensive than PCR reagents, RPA technology is equivalent in that results can be visualized by gel electrophoresis or with a fluorescent probe with greater sensitivity; and it is superior to the common PCR-based assay in its versatility, speed, and lack of need for a highly purified RNA template. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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