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With increased global trade, accurate identification of biosecurity pests considered to be significant risks to agriculture is critical. At present, there is a need for molecular aids to improve nematode diagnostics. In classical nematode diagnostics, a trained person who has acquired many years experience and knowledge of nematode morphology is required. To train a qualified nematode taxonomist takes time, and few experienced personnel are available in Australia. This research is part of a project to develop new strategies to identify biosecurity nematode pests that will contribute to safeguarding Australian agriculture from incursion of nematode pests.
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biosecurity built on science
Using ‘anti-primer’ technology for
nematode diagnostics
Matthew N. H. Tan PhD Candidate
Cooperative Research Centre for National Plant Biosecurity
biosecurity built on science
What are nematodes? - Microscopic worms
Different types of nematodes
The target organism…
Migratory Sedentary Ectoparasite Endoparasite Ectoparasite Endoparasite
Dagger nematode
Root lesion nematode
Ring nematode
Cyst nematode
Plant parasitic nematode Free living nematode Animal parasitic nematode
biosecurity built on science
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
Background…
biosecurity built on science
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
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 - 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
http://www.daff.gov.au/aqis/about/public-awareness/education/fact-sheets/cargo
Why conduct this study?
biosecurity built on science
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 high-throughput assay to improve the nematode diagnostics - Identify more species - Better accuracy - Faster - Cheaper
Aims:
biosecurity built on science
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
biosecurity built on science
Concept of ‘anti-primer’…
5’ 3’ 5’ 3’
5’ 3’
Unbound primer
5’ 3’
PCR product synthesis
Anti-primer Anchor 17 bp
Specific primer 24 bp
5’ 3’
Anti-primer 5’ 3’
°C
5’ 3’ A
C G T T G G C C A A
A
nematode DNA
C C C C A A A G
5’ 3’
5’ 3’ A C G T T G C C A A G A A A A C C G C C
Reverse primer
5’ 3’
biosecurity built on science
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
biosecurity built on science
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)
Steps… Pratylenchus spp.
biosecurity built on science
qPCR…Root lesion nematodes
Specific species primer
Species-specific primer with ‘anti-primer’ binding sequence
F R
F R
biosecurity built on science
qPCR…Root lesion nematodes
Multiplex using specific primer with ‘anti-primer’ binding site sequence
Multiplex system with FAM label attached
F R F R F R
Pn
Pp Pt
F R F R F R
Pn
Pp Pt *
biosecurity built on science
qPCR…Root lesion nematodes
Species-specific fluorescence labeled primer on specific template
F R F R F R
Pn
Pp Pt *
*
*
Multiplex using single template with different fluorescence labeled specific primer
F R F R F R
Pn
Pp Pt *
*
*
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
qPCR…Root lesion nematodes
Multiplex system – combination of different templates with 3 sets of species-specific primers
F R F R F R
Pn
Pp Pt *
*
*
Each sample with 3 species-specific primers
biosecurity built on science
‘Anti-primer’ multiplexing qPCR of Pn and Pp template using species-specific primers
Pn Channel (Yellow)
Pp Channel (Red)
Pt Channel (Green)
biosecurity built on science
‘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)
biosecurity built on science
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
Discussion…
biosecurity built on science
• To further develop a deep multiplex qPCR based on ‘Anti-primer’ technology
• Combine with rapid soil extraction method
• To submit my thesis
The next step…
biosecurity built on science
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)
For more information, please email [email protected]
Acknowledgements:
Cereal cyst nematode
