European Journal of Plant Pathology

, Volume 120, Issue 2, pp 177–188

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

  • Edson Bertolini
  • Aranzazu Moreno
  • Nieves Capote
  • Antonio Olmos
  • Ana de Luis
  • Eduardo Vidal
  • Jordi Pérez-Panadés
  • Mariano Cambra
Full Research Paper

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.

Keywords

Acquisition period CTV Tissue print-ELISA Tissue-print and squash real-time RT-PCR Viral titre 

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Copyright information

© KNPV 2007

Authors and Affiliations

  • Edson Bertolini
    • 1
  • Aranzazu Moreno
    • 1
  • Nieves Capote
    • 1
  • Antonio Olmos
    • 1
  • Ana de Luis
    • 1
  • Eduardo Vidal
    • 1
  • Jordi Pérez-Panadés
    • 1
  • Mariano Cambra
    • 1
  1. 1.Virología e Inmunología, Centro de Protección Vegetal y BiotecnologíaInstituto Valenciano de Investigaciones Agrarias (IVIA)MoncadaSpain

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