New technologies for monitoring plant viruses, vectors and viroids Sharon van Brunschot, PhD Candidate, CRCNPB & The University of Queensland, Australia
Research location:
Plant Research International, Wageningen University & Research Centre, The Netherlands
Supervisors:
Dr René van der Vlugt (WUR) Dr Jan Bergervoet (WUR)
biosecurity built on science Cooperative Research Centre for National Plant Biosecurity
Collaborative research project Plant Research International (Netherlands) & CRCNPB (Australia) Begomoviruses & Bemisia tabaci (whitefly) vectors of the TYLCD complex Tomato yellow leaf curl virus Tomato yellow leaf curl Sardinia virus B. tabaci Middle East Asia Minor 1 (B biotype)
Together, develop new multiplexed detection technology
B. tabaci Mediterranean (Q biotype)
Viroid species of the genus Pospiviroid (family Pospiviroidae) Potato spindle tuber viroid Tomato apical stunt viroid
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Luminex nucleic acid bead-based arrays
Recent uptake and adoption in clinical diagnostics
High throughput; very high multiplexing capability (up to 100 targets) & flexibility
Assay design simple, similar to qPCR
TAG
Target-specific primer extension product
anti-TAG
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Luminex xTAG bead suspension arrays Workflow = approximately 5 hours Plant or whitefly nucleic acid extraction
Sorting & detection
Multiplex PCR\RT-PCR & purification
Multiplex TSPE (target specific primer extension) with biotin dCTP incorporation
Hybridization of bead array with TSPE products
Addition of SAPE conjugate (streptavidin-phycoerythrin)
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Hierarchical assay design 7-plex begomovirus/whitefly bead array Sample
Solanaceae plant internal control
Begomovirus universal
β-satellite universal
B. tabaci universal 18S / ITS1 junction
FLORICAULA/LEAFY gene orthologue
TYLCV-IL specific
B. tabaci MEAM1 “B biotype”
B. tabaci MED “Q biotype”
mtCOI gene
mtCOI gene
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Hierarchical assay design 11-plex Pospiviroid bead array Sample
Plant internal control
Pospiviroid universal
Plant mitochondrial NADH dehydrogenase gene (nad5)
All species except Columnea latent viroid
Columnea latent viroid (CLVd) Citrus exocortis viroid (CEVd)
Iresine viroid (IrVd)
Tomato chlorotic dwarf viroid (TCDVd)
Potato spindle tuber viroid (PSTVd)
Tomato planta macha viroid (TPMVd)
Tomato apical stunt viroid (TASVd)
Pepper chat fruit viroid (PCFVd)
Chrysanthemu m stunt viroid (CSTVd)
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Results: Begomovirus-whitefly array
• Specificity assessed using sequence characterized, virus-infected plant samples • 100 % specificity
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Results: Begomovirus-whitefly array
• Specificity assessed using sequence characterized whitefly specimens (some viruliferous) • 100 % specificity
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Results: Pospiviroid array
• Specificity assessed using sequence characterized viroid-infected plant samples •100 % specificity
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New statistical method for threshold setting
• Thresholds calculated for each bead set based on a non-parametric, data-driven statistical procedure biosecurity built on science
Results: Begomovirus-whitefly array
Cloning and sequencing revealed B. tabaci Asia II species (K, P, ZHJ2 biotypes)
• PepLCIV positive for Beg & TOFL (Solanaceae internal control) bead sets • CLCuMV positive for Bem18S, Beta & Beg bead sets
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Sensitivity of Luminex bead array compared with qPCR TY Bead set, multiplexed Luminex array
TYLCV-IL specific singleplex qPCR
• Ten-fold dilution series of TYLCV-IL infected tomato gDNA extract • Limit of detection equivalent to qPCR = 204 fg DNA biosecurity built on science
Reproducibility over ten independent tests
• Highly reproducible results over independent tests (low variability of MFI readings, small standard deviation).
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Summary of results
7-plex bead array for the detection of begomoviruses and B. tabaci species
Starting material can be either virus infected plant or insect vector
11-plex bead array for the detection of all species with the genus Pospiviroid
Internal control assays to exclude false negatives
100% specificity
Sensitivity equivalent to qPCR
Proven reproducibility over ten independent tests
Novelty and outcomes of research
First application of multiplexed bead arrays for plant virus, vector and viroid nucleic acid-based diagnostics
New statistical methods for setting thresholds for positivity
Ready for use in Australia and Europe for routine indexing
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Thank you
For more information, please email sharon.vanbrunschot@uqconnect.edu.au
Acknowledgements
Dr René van der Vlugt (WUR)
Dr Jan Bergervoet (WUR)
Dr Daniel Pagendam (CSIRO Mathematics, Informatics and Statistics)
Dr Andrew Geering (QAAFI)
Dr André Drenth (QAAFI)
Dr John Thomas (QAAFI)
Marjanne de Weerdt (WUR)
Dr Paul De Barro (CSIRO Ecosystem Sciences)
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