Potato viruses such as Potato virus Y (PVY) cause diseases that affect potato quality and thus damage potato production worldwide. Current tests for viral infection use double-antibody sandwich enzyme linked immunosorbent assays (DAS ELISA) or reverse transcription polymerase chain reaction (RT-PCR)/real-time RT-PCR. Despite many advantages, these assays have a number of drawbacks that affect cost and time of diagnosis. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) allows fast detection of target RNA. Here, we developed a closed-tube real-time RT LAMP assay for fluorescent detection of PVY. Specific RT-LAMP primers were designed to target the conserved region of the sequence encoding the PVY coat protein. The assay was specific and facilitated sensitive PVY detection in a single tube at 65°C. The time-topositive values depended on the PVY concentration in tested samples. The effectiveness of RT LAMP in testing field-grown plants compared favorably with DAS ELISA and RT-PCR; under the tested conditions, RT-LAMP was about 1000-fold more sensitive than DAS ELISA and lateral flow assay (LFA) and about 10-fold more sensitive than RT PCR. Thus, this fluorescent RT-LAMP assay has great potential for routine detection of PVY. 1000 veces más sensible que DAS ELISA y que el ensayo de flujo lateral, y como 10 veces más sensible que RT-PCR. La efectividad de RT-LAMP al probarla en plantas en el campo se comparó favorablemente con DAS ELISA y RT-PCR. En consecuencia, presenta un gran potencial para detección de rutina del PVY.

In Poland, it is mandatory to index seed tubers for Potato virus Y (PVY), Potato virus M (PVM) and Potato leafroll virus (PLRV). Currently, the incidence of viral infection in seed tubers is determined by grow-out test. Direct testing of tubers after harvest by reverse transcription polymerase chain reaction (RT-PCR) can be beneficial but its application is so far hampered by the high cost of commercial kits for RNA isolation and poor usability of manual methods for routine diagnostics. In this work, we compare several commercial kits with the silica capture RT-PCR (SC-RT-PCR) for direct detection of PVY. The silica capture of RNA in conjunction with optimized PCR conditions facilitates quick and sensitive detection of PVY and offers cost effective and reliable alternative to commercial kits.Resumen En Polonia, los tubérculos-semilla son analizados obligatoriamente para el virus Y de la papa (PVY), virus M de la papa (PVM) y el enrollamiento de la hoja (PLRV). Actualmente, la incidencia de infección viral en tubérculo-semilla se determina por pruebas durante el crecimiento. Las pruebas directas de tubérculos después de la cosecha con la reverso-transcripción por la reacción en cadena de la polimerasa (RT-PCR) pudiera ser benéfica, pero su aplicación se limita por el alto costo de juegos comerciales para el aislamiento de ARN y por el pobre uso de métodos manuales para diagnósticos de rutina. En este trabajo comparamos varios juegos comerciales con la efectividad de costos de captura de sílice RT-PCR para detección directa de PVY. La sensibilidad en la detección del PVY en ARN preparado por captura de sílice fue igual o mayor que en ARN aislado usando juegos comerciales. El ARN por captura de sílice junto con condiciones óptimas de PCR facilita la detección rápida y sensible de PVY y ofrece una alternativa efectiva, en costos y confiabilidad, a los juegos comerciales.

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.

Procedures of separation of virus particles from a plant material are multistage. Furthermore often they are difficult in terms of methodology and require use of expensive, highly specialist equipment and yield of separation is often low. The antigen obtained is often degraded and contains admixtures of other proteins. Therefore, generation of high quality and specificity antibodies based on such antigen is very difficult and quality of the antibodies has impact on reliability, sensitivity and unambiguity of results of immunodiagnostic tests (e.g. ELISA) that are currently conventionally used to detect vegetable viruses. In this study three conventionally-performed methods of separation of potato virus Y (PVY) were compared and a method of separation based on membrane chromatography, as an alternative separation technique, has been presented. It has been demonstrated that in proper process conditions good quality virus preparation can be obtained.

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