Development of quantitative real-time RT-PCR for the detection and quantification of Peach latent mosaic viroid

Abstract

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 real-time 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.