Rapid and Sensitive Detection of Tomato Brown Rugose Fruit Virus in Tomato and Pepper Seeds by Reverse Transcription Loop-Mediated Isothermal Amplification Assays (Real Time and Visual) and Comparison With RT-PCR End-Point and RT-qPCR Methods
| Autor: | Giuseppe Parrella, Daniele Da Lio, Giovanni Cappellini, Linda Bartolini, Chiara Salemi, Alessandra Panattoni, Domenico Rizzo |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
Microbiology (medical) tomato brown rugose fruit virus RT-LAMP assay seed testing Loop-mediated isothermal amplification Biology 01 natural sciences Microbiology Virus 03 medical and health sciences seeds contamination Pepper Methods media_common.cataloged_instance European union Reverse Transcription Loop-mediated Isothermal Amplification 030304 developmental biology media_common Detection limit 0303 health sciences virus diagnostic fungi food and beverages QR1-502 eye diseases Horticulture Real-time polymerase chain reaction Seed testing 010606 plant biology & botany |
| Zdroj: | Frontiers in Microbiology Frontiers in microbiology (2021). doi:10.3389/fmicb.2021.640932 info:cnr-pdr/source/autori:Rizzo D., Da Lio D., Panattoni A., Salemi C., Cappellini G., Bartolini L., Parrella G./titolo:Rapid and Sensitive Detection of Tomato Brown Rugose Fruit Virus in Tomato and Pepper Seeds by Reverse Transcription Loop-Mediated Isothermal Amplification Assays (Real Time and Visual) and Comparison With RT-PCR End-Point and RT-qPCR Methods/doi:10.3389%2Ffmicb.2021.640932/rivista:Frontiers in microbiology/anno:2021/pagina_da:/pagina_a:/intervallo_pagine:/volume Frontiers in Microbiology, Vol 12 (2021) |
| ISSN: | 1664-302X |
| DOI: | 10.3389/fmicb.2021.640932 |
| Popis: | Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay [reverse transcription loop-mediated isothermal amplification (RT-LAMP)] and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/μl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-point RT-PCR diagnostic methods. In artificially contaminated seeds, the proposed LAMP assays detected ToBRFV in 100% of contaminated seed lots, for up to 0.025–0.033% contamination rates in tomato and pepper, respectively. Our results demonstrate that the proposed LAMP assays are simple, inexpensive, and sensitive enough for the detection of ToBRFV, especially in seed health testing. Hence, these methods have great potential application in the routine detection of ToBRFV, both in seeds and plants, reducing the risk of epidemics. |
| Databáze: | OpenAIRE |
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