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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

biosecurity built on science


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

biosecurity built on science


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)

biosecurity built on science


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

biosecurity built on science


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)

biosecurity built on science


Results: Begomovirus-whitefly array

• Specificity assessed using sequence characterized, virus-infected plant samples • 100 % specificity

biosecurity built on science


Results: Begomovirus-whitefly array

• Specificity assessed using sequence characterized whitefly specimens (some viruliferous) • 100 % specificity

biosecurity built on science


Results: Pospiviroid array

• Specificity assessed using sequence characterized viroid-infected plant samples •100 % specificity

biosecurity built on science


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

biosecurity built on science


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).

biosecurity built on science


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

biosecurity built on science


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)

biosecurity built on science


New technologies for plant biosecurity: bead arrays for multiplexed detection of plant viruses