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Publication datasheet
Title:
Development of quantitative real-time RT-PCR for the detection and quantification of Peach latent mosaic viroid
Authors:
Luigi, M.; Faggioli, F.
Year:
2011
Languages:
ENG, eng
Journal:
European Journal of Plant Pathology
Kind of publication:
Elettronico
Location:
Editor:
Springer
Abstract in Italian:
Abstract in English:
Peach latent mosaic viroid (PLMVd) is a damaging pathogen for peach, its detection being of critical importance to both sanitary and certification programs worldwide. Here we report a quantitative realtime reverse transcription polymerase chain reaction assay (qRT-PCR) based on TaqMan® chemistry to improve the diagnosis of this pathogen. Critical to this approach is the design of a specific set of primers and of a probe, and the use of a suitable extraction method proving a reliable, sensitive and specific PLMVd diagnosis. More specifically, the sensitivity was evaluated using 12 ten-fold dilution series of an in vitro transcript of the entire PLMVd genome as a standard for quantification in infected samples. The assay detected up to 1 fg of target RNA. The protocol was also evaluated for its specificity using healthy peach, apricot, plum and pear controls and non-target viroids (Apple scar skin viroid, Hop stunt viroid, Pear blister canker viroid, Apple dimple fruit viroid and Potato spinder tuber viroid). None of the non-target or healthy analysed samples reacted in qRT-PCR. The efficiency and accuracy of the method was evaluated using different PLMVd peach isolates as templates, including three ‘calico’ variants: a strong signal was obtained from all of them. Finally, five RNA extraction methods were compared and evaluated to choose the best for detection and quantification of PLMVd by the qRT-PCR protocol.

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