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Quantitative detection of Citrus tristeza virus in plant tissues and single aphids by real-time RT-PCR

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Abstract

TaqMan real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) using purified RNA targets or coupled to tissue-print and squash procedures was developed to detect and quantify Citrus tristeza virus (CTV) RNA-targets in plant tissues and in single aphids. With this method all CTV isolates tested from different hosts and origins were detected. The sensitivity of conventional real-time RT-PCR was 1,000 times higher than immunocapture (IC)-RT-nested PCR and 106 times higher than enzyme linked immunosorbent assay (ELISA). The quantitation limit ranged from 1.7 × 102 to 1.7 × 109 transcript copies. The estimated number of CTV RNA-targets detected in different organs of a CTV-infected tree ranged from 4.5 × 105 to 6.5 × 108 copies when purified RNA was used as template and from 1.9 × 104 to 3.7 × 106 when tissue-printed material was used. In single squashed aphids the number of copies ranged from 4.73 × 103 to 1.23 × 105. Reliable quantitation of CTV targets present in infected plant material or acquired by single aphid species, was achieved with tissue-print and squash procedures combined with real-time RT-PCR, both of which do not require extraction procedures or nucleic acid purification.

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Acknowledgements

This work was supported by the INIA RTA2005-00190 (IVIA 1606) project. E. Bertolini and A. Moreno are recipients of a Juan de la Cierva contract from Ministerio de Educación y Ciencia of Spain. A. de Luis was the recipient of a fellowship grant from IVIA (Generalidad Valenciana). Dr. E. Carbonell assisted in statistical analysis. Dr. N. Duran-Vila critically read the manuscript. English text was revised by F. Barraclough.

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Authors and Affiliations

  1. Virología e Inmunología, Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera Km 5, 46113, Moncada, Valencia, Spain

    Edson Bertolini, Aranzazu Moreno, Nieves Capote, Antonio Olmos, Ana de Luis, Eduardo Vidal, Jordi Pérez-Panadés & Mariano Cambra

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  1. Edson Bertolini
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  2. Aranzazu Moreno
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  3. Nieves Capote
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  4. Antonio Olmos
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  5. Ana de Luis
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  6. Eduardo Vidal
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  7. Jordi Pérez-Panadés
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  8. Mariano Cambra
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Correspondence to Mariano Cambra.

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Bertolini, E., Moreno, A., Capote, N. et al. Quantitative detection of Citrus tristeza virus in plant tissues and single aphids by real-time RT-PCR. Eur J Plant Pathol 120, 177–188 (2008). https://doi.org/10.1007/s10658-007-9206-9

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  • DOI: https://doi.org/10.1007/s10658-007-9206-9

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