Abstract
Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.
Highlights
Potato virus Y (PVY) is the type member of the genus Potyvirus, family Potyviridae [9]
We describe a streamlined magnetic capture version of the reverse transcription loop-mediated amplification (MC-RT-LAMP) assay that we optimized for fast detection
The high specificity of the RT-LAMP method relies heavily on the primer sets designed for the amplification of the targeted region
Summary
Potato virus Y (PVY) is the type member of the genus Potyvirus, family Potyviridae [9]. Many procedures for the detection of PVY in potato leaves and tubers have been reported [3, 6, 13,14,15,16] Due to their easy adaptation to automation, their high specificity, and their ability to detect viral strains in a single reaction, real-time-RT-PCR-based assays appear to be ideal successors of the double sandwich enzyme-linked immunosorbent assay (DAS-ELISA) that is routinely used in the grow-out test of dormant tubers for the diagnosis of potato viruses for seed certification [3]. PVY isolate 12/94 (PVYNTN) was used in all experiments
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