title: 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.
authors.affiliation: Plant Biosecurity Cooperative Research Centre, Bruce, Australian Capital Territory, Australia ; School of Agriculture and Food Sciences, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Sharon L
authors.initials: SL
authors.lastname: van Brunschot
authors.affiliation: Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Jan H W
authors.initials: JH
authors.lastname: Bergervoet
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation Mathematics, Informatics and Statistics, Dutton Park, Queensland, Australia.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Pagendam
authors.affiliation: Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Marjanne
authors.initials: M
authors.lastname: de Weerdt
authors.affiliation: Plant Biosecurity Cooperative Research Centre, Bruce, Australian Capital Territory, Australia ; Centre for Plant Science, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Andrew D W
authors.initials: AD
authors.lastname: Geering
authors.affiliation: Centre for Plant Science, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: André
authors.initials: A
authors.lastname: Drenth
authors.affiliation: Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: René A A
authors.initials: RA
authors.lastname: van der Vlugt
mesh_terms: D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010942:Plant Viruses / Q000145:classification / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014772:Viroids / Q000145:classification / Q000235:genetics*