Development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens

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.

Figure 1. Flow diagram depicting the five steps of the Luminex MagPlex-TAG pospiviroid array method.

Figure 2. Specificity of the Luminex MagPlex-TAG pospiviroid array when screened against sequence-characterized pospiviroid isolates.

Performance of the 11-plex bead-based array when screened against a large panel of single and mixed infections of sequence-characterized pospiviroid isolates, obtained from natural infections of a variety of host plants (with mixed infections simulated). Data for healthy tomato (uninfected control) and blank (no template control) samples are included for reference. Asterisks denote mean natural log (ln) median fluorescence intensity (MFI) values that exceed the threshold for that assay within the multiplexed bead-based array.

Figure 3. Reproducibility of the Luminex MagPlex-TAG pospiviroid array over ten independent tests.

One sample of Potato spindle tuber viroid (isolate #3077695) was tested in ten independent reactions over several days. Mean natural log (ln) median fluorescence intensity (MFI) values are plotted; error bars show plus or minus (±) one standard deviation. The horizontal bars plotted on each of the bars shows the detection threshold obtained from our kernel density estimation method, for each assay of the array. The small standard deviation of the ln(MFI) values for positively reacting assays in the array (PSTVd, PospUni and PlantIC) demonstrate the high level of precision of the multiplexed bead-based array.