Rapid detection of tomato spotted wilt virus by real-time RT-LAMP and in-field application
Tomato spotted wilt virus (TSWV) is considered one of the most threatening viruses worldwide for diferent economically important agricultural crops. In this scenario, it is important to perform an early detection by laboratory tests to prevent TSWV spread. A rapid and sensitive TSWV detection protoc...
| Autores principales: | , , , , , , , , |
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| Formato: | article |
| Lenguaje: | Inglés |
| Publicado: |
Springer
2024
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| Materias: | |
| Acceso en línea: | https://hdl.handle.net/20.500.11939/8833 https://link.springer.com/article/10.1007/s42161-024-01613-3 |
| Sumario: | Tomato spotted wilt virus (TSWV) is considered one of the most threatening viruses worldwide for diferent economically important agricultural crops. In this scenario, it is important to perform an early detection by laboratory tests to prevent TSWV spread. A rapid and sensitive TSWV detection protocol based on real time reverse transcription loop-mediated isothermal amplifcation (RT-LAMP) assay was developed in this work, also using cost-efective and simplifed sample preparation procedure, to assess the suitability of the RT-LAMP assay in feld conditions on tomato and pepper samples. A set of six primers was designed within the nucleotide sequence region coding for the nucleocapsid protein (N) of segment S, targeting a 220-nucleotide sequence. Sensitivity, specifcity, accuracy, and in-feld application of the real-time RT-LAMP assay were evaluated. The developed real-time RT-LAMP assay proved to be one thousand and one hundred times more sensitive than end-point RT-PCR and real-time RT-PCR methods, respectively, detecting a total of 9.191 × 101 genome copies as minimum target, and no cross-reactivity were detected with other viruses belonging to Tospoviridae and Bromoviridae families used as outgroup. In addition, the in-feld application of the assay using the rapid sample preparation gave adequate and reliable results within 60 minutes, with an acceptable reaction delay when compared to canonical RNA extraction. The in-feld analyses showed an increase of TSWV-positive samples (37%) detection compared
with end-point RT-PCR and real-time RT-PCR (32% and 29%, respectively), particularly on asymptomatic samples, confrming that the real-time RT-LAMP assay can be implemented as a routine test both in-feld and laboratory conditions as a rapid and sensitive technique for TSWV detection. |
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