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

Authors:Bertolini, E.
Moreno, Aránzazu
Capote, Nieves
Olmos, A.
Luis, Ana de
Vidal, E.
Pérez Panadés, Jordi
Cambra, Mariano
Keywords:Acquisition period, Tissue print-ELISA, CTV, Tissue print and squash real time RT-PCR, Viral titre
Issue Date:2008
Citation:European Journal of Plant Pathology 120: 177-188 (2008)
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 realtime 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 CTVinfected 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.
Description:12 páginas, ilustraciones y tablas estadísticas
Publisher version (URL):http://dx.doi.org/10.1007/s10658-007-9206-9
Appears in Collections:(ICA) Artículos

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