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| Content Provider | Springer Nature Link |
|---|---|
| Author | Przewodowska, Agnieszka Zacharzewska, Bogumiła Chołuj, Joanna Treder, Krzysztof |
| Copyright Year | 2015 |
| Abstract | 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-to-positive 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.Los virus de la papa causan enfermedades que reducen enormemente la calidad. Entre ellos, el virus Y de la papa (PVY) es el más importante en la producción de papa en todo el mundo. En la actualidad la incidencia de la infección viral en papa se prueba mediante DAS ELISA o RT-PCR/RT-PCR de tiempo real. A pesar de muchas ventajas, estos ensayos tienen un número de dificultades que afectan el costo y el tiempo del diagnóstico viral. Una amplificación térmica de transcripción reversa mediada por enroscamiento (RT-LAMP) es un método novedoso que permite detección rápida del ARN objetivo. En este trabajo se desarrolló un ensayo en tubo cerrado de RT-LAMP de tiempo real para detección fluorescente del PVY. Se diseñaron iniciadores específicos RT-LAMP apuntando a la región conservada de la secuencia que codifica para la cubierta proteica. El ensayo fue específico y facilitó la sensibilidad de detección del PVY en tubo individual a 65 °C. Los valores del tiempo al positivo dependieron de la concentración del PVY en las muestras probadas. Bajo las condiciones de prueba, RT-LAMP fue cerca de 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. |
| Starting Page | 303 |
| Ending Page | 311 |
| Page Count | 9 |
| File Format | |
| ISSN | 1099209X |
| Journal | American Potato Journal |
| Volume Number | 92 |
| Issue Number | 3 |
| e-ISSN | 18749380 |
| Language | English |
| Publisher | Springer US |
| Publisher Date | 2015-02-11 |
| Publisher Institution | Potato Association of America |
| Publisher Place | New York |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Potato virus Y Reverse transcription loop-mediated isothermal amplification assay Potato Plant Sciences Agriculture Plant Genetics & Genomics Plant Breeding/Biotechnology Plant Pathology |
| Content Type | Text |
| Resource Type | Article |
| Subject | Plant Science Agronomy and Crop Science |
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