Using ‘anti-primer’ technology for nematode diagnostics Matthew N. H. Tan PhD Candidate
biosecurity built on science Cooperative Research Centre for National Plant Biosecurity
The target organism‌  What are nematodes? - Microscopic worms
 Different types of nematodes Plant parasitic nematode Animal parasitic nematode
Migratory
Free living nematode
Sedentary
Ectoparasite
Endoparasite
Ectoparasite
Endoparasite
Dagger nematode
Root lesion nematode
Ring nematode
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Background… Global agricultural production losses caused by plant parasitic nematodes are estimated at USD$157 Billion (Abad et al., 2008) In Australia, the annual loss is estimated at AUD$600 million (Hodda and Cook, 2009, Murray and Brennan, 2009)
Classical taxonomy is important and there are some molecular techniques used for identification/detection but these methods are time consuming For quarantine purposes, techniques which enable the detection and identification of quarantine pests within a short period of time are required
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An example of the worldwide dispersion of a plant parasitic nematode (Potato cyst nematode) 1690s
(Modify from Brodie, B. B. et al. 1993) biosecurity built on science
Why conduct this study? To reduce identification time compared with classical taxonomy -
Reduce detection and identification time from weeks or days to hours or minutes
To develop new methods of nematode diagnostics -
http://www.daff.gov.au/aqis/about/ public-awareness/education/factsheets/cargo
For identification of possible future incursions of plant pests, ‘anti-primer’ technology is a potentially useful addition to available techniques
To investigate use of ‘anti-primer’ technology for identification of nematodes -
Quarantine materials require fast and accurate detection and identification
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Aims: To develop and validate ‘anti-primer’ technology for identification of nematode species - Internal transcribed spacer rDNA
To test the potential of this technology as a highthroughput assay to improve the nematode diagnostics -
Identify more species Better accuracy Faster Cheaper
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Background of anti-primer qPCR technology
‘Anti-primer’ technology has been used in a clinical setting, e.g. cancer research
First use in plant pathology
A modified qPCR set up, -
with a fluorescently labelled forward primer and an ‘anti-primer’ with a quencher of fluorescence
‘Anti-primer’ is not involved in DNA amplification but quenches free fluorescent forward primer
Potential high-throughput assay using qPCR to detect different nematodes
Design of ‘anti-primer’ provide better quenching of fluorescence
Greater sensitivity due to low background
Specific detection
Results can be obtained in less than 2hrs
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Concept of ‘anti-primer’… 5’
nematode DNA
3’
Anti-primer Anchor 17 bp
TA AT GCACA GG A AC C CC C G A
Specific primer 24 bp
5’
3’
3’ 3’
5’ 3’ACGGTA ATGCACA A ACC GCC
Reverse primer
5’
5’ 3’
5’ 5’
Anti-primer 3’
5’
3’
°C
Unbound primer PCR product synthesis 5’
3’
5’
3’
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qPCR…
different fluorescent label
Channel
Excitation (nm)
Detection (nm)
Examples of fluorophores detected
Blue
365±20
460±20
Edans
Green
470±10
510±5
FAM
Yellow
530±5
557±5
JOE
Orange
585±5
610±5
ROX
Red
625±5
660±10
Cy5
Crimson
680±5
712 high pass Alexa Fluor 680
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Steps…
Pratylenchus spp.
Root lesion nematodes (RLN)
Pratylenchus neglectus (Aus/WA DAFWA) P. penetrans (Aus/WA DAFWA) P. thornei (Aus/Vic DAFWA) P. zeae (Aus/Qld)
DNA extraction and qPCR primer design on ITS region -
Specific primer for each species (~100bp)
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qPCR…Root lesion nematodes Specific species primer F R
Species-specific primer with ‘anti-primer’ binding sequence F R
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qPCR…Root lesion nematodes Multiplex using specific primer with ‘anti-primer’ binding site sequence Pn
F
Pp
F
R
Pt
F
R R
Multiplex system with FAM label attached Pn
F
Pp
F
R
Pt * F
R R biosecurity built on science
qPCR‌Root lesion nematodes Species-specific fluorescence labeled primer on specific template Pn* F Pp* F Pt * F
R R R
Multiplex using single template with different fluorescence labeled specific primer Pn* F Pp* F Pt * F
R R R Each sample with 3 species-specific primers biosecurity built on science
‘Anti-primer’ multiplexing qPCR of single template using species-specific primers Pn DNA template
Pp DNA template
Pt DNA template
biosecurity built on science
qPCR‌Root lesion nematodes Multiplex system – combination of different templates with 3 sets of species-specific primers
Pn* F Pp* F Pt * F
R R R Each sample with 3 species-specific primers
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‘Anti-primer’ multiplexing qPCR of Pn and Pp template using species-specific primers Pn Channel (Yellow)
Pp Channel (Red)
Pt Channel (Green)
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‘Anti-primer’ multiplexing qPCR of Pn and Pt template using species-specific primers Pn Channel (Yellow)
Pp Channel (Red)
Pt Channel (Green)
biosecurity built on science
‘Anti-primer’ multiplexing qPCR of Pp and Pt template using species-specific primers Pn Channel (Yellow)
Pp Channel (Red)
Pt Channel (Green)
biosecurity built on science
‘Anti-primer’ multiplexing qPCR of Pn, Pp and Pt template using species-specific primers Pn Channel (Yellow)
Pp Channel (Red)
Pt Channel (Green)
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Discussion… Anti-primer • With different fluorescent labels, different species can be detected in a single PCR reaction • Can identify species using specific primers • Need to select primers to avoid possible cross reactions • Increases potential for nematode diagnostics • Cost effective since only 1 ‘anti-primer’ is needed and can standardise all reactions using this system • ITS region of most nematodes of biosecurity concern have been sequenced and new specific primers can be readily designed
The current anti-primer technology using qPCR can already be applied to address nematode biosecurity issues
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The next step… • To further develop a deep multiplex qPCR based on ‘Anti-primer’ technology • Combine with rapid soil extraction method • To submit my thesis
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Acknowledgements: I would like to thank the following -
CRC for National Plant Biosecurity Prof Michael Jones (Murdoch University) Dr Dave Berryman (Murdoch University) Dr Vivien Vanstone (DAFWA) Mrs Helen Hunter (DAFWA) Dr Kerrie Davies (Uni of Adelaide) Ms Jo-Anne Tan (SABC PhD student)
Cereal cyst nematode For more information, please email m.tan@crcplantbiosecurity.com.au
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