Abstract
Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.
Highlights
Viroids are the smallest plant pathogens known to date
The step of the multiplex array involved a highly specific multiplexed target specific primer extension step using a mix of 11 TSPE primers, during which biotin-dCTP was incorporated into the extension products
This study reports the development and validation of the 11-plex Luminex MagPlexTAG Pospiviroid array, a valuable tool for the simultaneous detection of all nine recognized viroid species in the genus Pospiviroid, from both crop and ornamental plant species
Summary
Viroids are the smallest plant pathogens known to date They are autonomously replicating, unencapsidated infectious RNAs that move systemically throughout an infected plant. Their circular, single-stranded genomes range in size from ,250–400 nucleotides (nt), and do not encode any proteins [1,2]. The genus Pospiviroid (family Pospiviroidae) contains ten recognized species: Chrysanthemum stunt viroid (CSVd), Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), Iresine viroid 1 (IrVd-1), Mexican papita viroid (MPVd), Pepper chat fruit viroid (PCFVd), Potato spindle tuber viroid (PSTVd), Tomato apical stunt viroid (TASVd), Tomato chlorotic dwarf viroid (TCDVd), and Tomato planta macho viroid (TPMVd) [3]. Recent research has demonstrated the conspecificity of MPVd and TPMVd, and their reclassification to a single species, namely TPMVd, has been proposed [4]
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