Abstract
Direct systems to process samples allow high-throughput testing or identification of Citrus tristeza virus (CTV) by the sensitive real-time reverse transcription coupled to polymerase chain reaction (RT-PCR) neither with extract preparation nor nucleic acid purification. Immobilized CTV targets are amplified from fresh sections of plant tissues or squashes of fresh or already caught vectors onto paper, nitrocellulose, or positively charged nylon membranes. The printed or squashed support can be stored or mailed at room temperature. These validated user-friendly methods are recommended by IPPC-FAO standard for CTV diagnosis, detection, and identification. The methods are safe, not under current quarantine regulations because they do not involve any risk of introduction of exotic CTV isolates or vectors and are discrete and useful for epidemiological studies or screening for large-scale analyses. In this chapter, tissue-printing and squashing capture methods for direct sample preparation without extract preparation or nucleic acid extraction and purification were coupled with validated real-time RT-PCR detection protocols based on TaqMan chemistry for CTV detection.
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Acknowledgment
This work was supported by Instituto Nacional de Investigaciones Agrarias (INIA) and Instituto Valenciano de Investigaciones Agrarias (IVIA) grants and for Plant Print Diagnostics SL agreements for the development and validation of direct methods of sample preparation. The methods were patented in Spain, EU: (1) “Procedure for targets preparation prior to PCR,” N° P9601155/6, May 17, 1996, by M. Cambra, A. Olmos, and MA Dasí (IVIA) and (2) “Procedure for direct and specific detection of ‘Candidatus Liberibacter spp’, Potato spindle tuber viroid, Citrus exocortis viroid and Peach latent mosaic viroid by immobilized targets and real-time PCR,” N° 2001001157, September 8, 2010 (IVIA and Fundecitrus), by E. Bertolini, M. Cambra, P. Serra, MM López, S. Lopes, N. Durán-Vila, J. Ayres, and JM. Bové. We are grateful to the authors and to the people of the Virology and Immunology Department team of Plant Protection and Biotechnology Centre of IVIA that participated in the research involved in this work over the last 20 years.
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Cambra, M., Vidal, E., Martínez, C., Bertolini, E. (2019). Tissue-Print and Squash Capture Real-Time RT-PCR Method for Direct Detection of Citrus tristeza virus (CTV) in Plant or Vector Tissues. In: Catara, A., Bar-Joseph, M., Licciardello, G. (eds) Citrus Tristeza Virus. Methods in Molecular Biology, vol 2015. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9558-5_5
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DOI: https://doi.org/10.1007/978-1-4939-9558-5_5
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