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

Sharon L van Brunschot; Jan H W Bergervoet; Daniel E Pagendam; Marjanne de Weerdt; Andrew D W Geering; André Drenth; René A A van der Vlugt

doi:10.1371/journal.pone.0084743

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

PLoS One. 2014 Jan 3;9(1):e84743. doi: 10.1371/journal.pone.0084743. eCollection 2014.

Authors

Sharon L van Brunschot¹, Jan H W Bergervoet², Daniel E Pagendam³, Marjanne de Weerdt², Andrew D W Geering⁴, André Drenth⁵, René A A van der Vlugt²

Affiliations

¹ Plant Biosecurity Cooperative Research Centre, Bruce, Australian Capital Territory, Australia ; School of Agriculture and Food Sciences, The University of Queensland, St Lucia, Queensland, Australia.
² Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands.
³ Commonwealth Scientific and Industrial Research Organisation Mathematics, Informatics and Statistics, Dutton Park, Queensland, Australia.
⁴ Plant Biosecurity Cooperative Research Centre, Bruce, Australian Capital Territory, Australia ; Centre for Plant Science, The University of Queensland, St Lucia, Queensland, Australia.
⁵ Centre for Plant Science, The University of Queensland, St Lucia, Queensland, Australia.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Multiplex Polymerase Chain Reaction / methods*
Plant Viruses / classification
Plant Viruses / genetics*
Reproducibility of Results
Sensitivity and Specificity
Viroids / classification
Viroids / genetics*

Grants and funding

This study was supported by a grant from Interreg “Healthy Greenhouse” (www.gezondekas.eu). The authors would like to acknowledge the support of the Plant Biosecurity Cooperative Research Centre (Project No. CRC60070), established and supported under the Australian Government's Cooperative Research Centres Program (http://www.crcplantbiosecurity.com.au). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.