Diagnostic kits for

identification & detection of nematodes

Renske Landeweert & Hans Helder
QBOL-EPPO meeting Haarlem
May 2012
New start up company:
Joined initiative of
Wageningen University & BLGG AgroXpertus
Founded December 2011
Based in Wageningen (NL)
Offering molecular diagnostics to agricultural labs
Nematodes cause US$ 80 billion damage annually
Phasing out of pesticides
Durable alternatives depend on accurate diagnostics
Problem
Nematodes are numerous & speciose, but possess few
informative morphological characters
Picture from Greg Tylka (Iowa State University)
Problem (continued)
Agricultural (inspection) laboratories need analyses:
More accurate
Less labour intensive
Up-scalable
More cost effective
Current situation
dr. Hans Helder
Laboratory of Nematology, Wageningen University
Sven van den Elsen, Paul Mooyman, Martijn Holterman
Solution
Single worm classical identification & digital high resolution pictures
Building a database that links morphological and molecular data
DNA extraction
DNA amplification
cloning into vector
DNA sequencing
Nematode DNA database
Nematode DNA wallpaper
GTGGTAAACTTCATCTAAGACTTAATATTGCCACGAGGCCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGTGAGGTG
Cl 1129+1626+1627 GTGGTAAACTTCATCTAAGACTTAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGTAAGGTG
Cl 1109+1622+1743 GTGGTAAACTTCATCTAAGACCTAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACATTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGTAAGGTG
clone1586+1587+1588 GTGGTAAACTTCATCTAAGACTCAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGTGAGGTG
clone1589+1590+1591 GTGGTAAACTTCATCTAAGACTTAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGTAAGGTG
clone1592+1593+1594 GTGGTAAACTTCATCTAAGACTTAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGTAAGGTG
clone1595+1596+159 GTGGTAAACTTCATCTAAGACTCAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGATAGAGTTAAAGAGGACGTGAAACCGGAGAGGTG
clone1598+1599+1600 GTGGTAAACTTCATCTAAGACTTAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGTAAGGTG
clone1601+1602+1603 GTGGTAAACTTCATCTAAGACTTAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGTAAGGTG
clone1607+1608+16 GTGGTAAACTTCATCTAAGACTTAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGTAAGGTG
Cl 1610+1611+1612 GTGGTAAACTTCATCTAAGACTCAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAAGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGAGAGGTG
Cl 1613+1614+1615 GTGGTAAACTTCATCTAAGACTCAATATTGCCACGAGACCGATAGCAAACAAGTACTGTGAAGGAAGGTTGCAAAGCACTTTGAAGAGAGAGTTAAAGAGGACGTGAAACCGGAGAGGTG
Proprietary database with 2,500 SSU rDNAs
covering all major terrestrial and freshwater
nematode groups (= haystack)
Specific DNA sequence signatures for most plant
parasitic nematodes (= needles)
Nematode DNA database
World-wide largest nematode DNA database.
. enabling clear detection
of needles in haystack
Nematode DNA database
World-wide largest nematode DNA database
Proprietary nematode DNA extraction method
Proprietary primers for plant pathogenic nematodes
Diagnostic Q-PCR tests (validated and in use)
We generated:
M.chit. DNA +
M.chit. primers
10
-6
10
-5
10
-5
10
-6
M.fal. DNA +
M.fal. primers
M.fal. DNA +
M.chit. primers
M.chit. DNA +
M.fal. primers
Specificity of Q-PCR tests (1)
Specificity of Q-PCR tests (2)
M.fal. DNA +
(M.chit. DNA +)
M.fal. primers
M.fal. M.chit.
10
-4
10
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10
-5
10
-5
10
-6
10
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10
-7
10
-5
10
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10
-5
Sensitivity of Q-PCR tests
Most accurate diagnostics of nematodes:
M.chitwoodi
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Aantal aaltjes per besmetting
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Mic. 2007
DNA 2007
M.minor
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1--10 10--100 100--1000 1000-100000
Aantal aaltjes per besmetting
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Mic. 2007
DNA 2007
#nematodes/infected sample
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#nematodes/infected sample
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microscope
molecular
Calibration line Chitwoodi
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20 22 24 26 28 30
Average Ct value (Q-PCR)
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Calibration line M.Fallax
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22 24 26 28 30 32
Average Ct value (Q-PCR)
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M. chitwoodi
1 40 nematodes
M. fallax
1 40 nematodes
Quantification with Q-PCR tests
Most technologies:
Molecular identification
in a pre-selected pool
- Barely up-scalable
- Not automatable
Our technology:
Molecular detection and
quantification without any
pre-selection
- Up-scalable
- Automatable
target
Main advantage of our Q-PCR tests:
Service laboratory: routine analyses of nematodes
> 40.000 soil samples / year for nematodes
> 250.000 Q-PCR tests / year (~ 20.000 samples)
Extensive use of Q-PCR tests
40.000 samples/year
20.000 samples/year 20.000 samples/year
Current routine workflow
Quantitative molecular tests (Q-PCR SybrGreen based):
Meloidogyne chitwoodi*, M. fallax*, M. minor, M. naasi, M. hapla
Globodera rostochiensis*, G. pallida*, G. artemisiae, G. achilleae
Ditylenchus dipsaci, D. destructor
Heterodera schachtii, H. betae/trifolii
Pratylenchus thornei, P. crenatus, P. penetrans, P. neglectus,
P. vulnus
Trichodorus spp., T. primitivus, T. similis, T. viruliferus
Paratrichodorus spp., P. teres, P. pachydermus, P. nanus,
P. anemones
Radopholus similis
..
+ ~20 families of non-plant parasitic nematodes
* in EPPO Standards
Currently in use
1) Diagnostic kits for nematodes:
DNA extraction & purification
Species identification
Species quantification
2) Dedicated system
(including Training, Support and Trouble shooting)
ClearDetections offers
Most accurate diagnostics of nematodes:
Simple protocol (monophasic)
Identical PCR program for all primers
Specific and sensitive
Objective and robust
Quantitative
No sequencing required
No fishing required
Easy to standardize
Europe: ClearDetections tests in EPPO Standards
(European Plant Protection Organisation)
ClearDetections offers
All primers have the same annealing temperature (63C):
Meloidogyne chitwoodi*, M. fallax*, M. minor, M. naasi, M. hapla
Globodera rostochiensis*, G. pallida*, G. artemisiae, G. achilleae
Ditylenchus dipsaci, D. destructor
Heterodera schachtii, H. betae/trifolii
Pratylenchus thornei, P. crenatus, P. penetrans, P. neglectus,
P. vulnus
Trichodorus spp., T. primitivus, T. similis, T. viruliferus
Paratrichodorus spp., P. teres, P. pachydermus, P. nanus,
P. anemones
Radopholus similis
..
+ ~20 families of non-plant parasitic nematodes
* in EPPO Standards
Ta: 63C
DNA extraction kit (single nematodes and communities)
Species identification kits for:
Meloidogyne chitwoodi & M. fallax
Globodera rostochiensis & G. pallida & G. tabacum
Pratylenchus (several species)
Heterodera (several species)
Ditylenchus dipsaci & D. destructor
Many more to follow: also fungi, bacteria, viruses
Our first kits
DNA extraction & purification
materials
Q-PCR primers for Control
DNA
Simple protocol
Q-PCR primers (SybrGreen)
for target species
Q-PCR primers (SybrGreen)
for all nematodes as control
Positive control DNA
(plasmid) of target species
Negative control DNA
(plasmid) of non-target
species
Simple protocol
Nematode identification kits
Website:
www.cleardetections.com
availabe from end of June 2012
YouTube movie: search for cleardetections
http://www.youtube.com/watch?v=LEKW2CbdkDc
More information, suggestions & contact:
info@cleardetections.com
Thank you!