Cor Schoen

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On-Site Confirmation and Monitoring

• Cor Schoen

29th Nov, 2010 Q-detect
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WP7. On-Site Confirmation and Monitoring

• Main objective To develop rapid, simple and
  reliable confirmatory and monitoring methods
  based on the detection of DNA

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Overview of work for Task 7
Task 7.1 DNA extractions Task 7.2 Generation of
protocols for isothermal, singleplex and
multiplex amplification of targets Different
isothermal amplification strategies will be
tested for on-site confirmation LAMP, NAIMA
and PLP-RCA. Task 7.3 Generation of devices for
multiplex detection on-site Task 7.4 Evaluation
of promising on-site detection systems for
monitoring This task may comprise the following
subtasks 1) evaluate selected robust DNA
extraction procedure for each pathosystem 2)
compare classical PCR to selected isothermal
methods 3) select optimal single-plex and
multiplex detection methods 4) establish one
protocol with strong focus on simplicity Task 7.5
Implementation of procedure on user-friendly
detection system
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Confirmation and Monitoring

• DNA/RNA extractions on different substrates to be
  performed in the field and/or on-site
• White flies viruses from traps
• Bacterial pathogens from plant material
• Potato pathogens from tuber and leaves

DNA/RNA Extraction Criteria - Easy -
Fast - Efficient - Cheap
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With respect to simplicity, speed and pricing,
purification of nucleic acid (RNA and DNA) based
on lateral flow devices would be ideal.
Boonham et al 2008
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Boonham et al 2008
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Boonham et al 2008
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• Conclusion extractions
• Qiagen DNeasy/RNeasy extraction kids have been
  successfully used for most of the described
  targets in WP7

• LFD based DNA/RNA extractions
• Easy to perform
• Fast
• Applicable for different substrates
• (RNA and DNA viruses has to be tested)
• Recovery has to be improved

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To validate LAMP, NAIMA and PLP-RCA, different
TaqMan assays were developed as the golden
standard for target detection
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Confirmation and Monitoring

• DNA/RNA extractions on different substrates to be
  performed in the field and/or on-site
• White flies viruses from traps
• Bacterial pathogens from plant material
• Potato pathogens from tuber and leaves
• Monitoring
• Isothermal multiplex and simplex amplification
• LAMP (Optisense)
• NAIMA ?
• Confirmation
• Isothermal multiplex and simplex amplification
• LAMP (Optisense)
• PLP in combination with RCA (Qlinea)

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Extraction
DNA / RNA
React.Mix
First line screening (monitoring via fast semi
specific method)
-

?
Second line screening (confirmation via target
specific detection methods)
Specific target detection
Real positive? Confirmation with another
technology
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• Monitoring (first line screening)
• Isothermal multiplex and simplex amplification
• Easy
• Fast
• Sensitive
• Applicable for DNA and RNA

?
- PCR - PLP - RCA - NAIMA - LAMP
?
?
?
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Isothermal amplification
LAMP Loop mediated Amplification
- Amplification at a constant temperature (65oC)
using a DNA polymerase with strand displacement
reaction - Amplification and detection of the
target can be completed in a single step. - High
amplification efficiency, with DNA being
amplified 109- 1010 times in 15- 60 minutes
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LAMP characteristics (summary) There is no
need for a step to denature double stranded into
a single stranded form. The whole
amplification reaction takes place continuously
under isothermal conditions. Amplification
can be done with RNA templates following the same
procedure as with DNA templates, simply through
the addition of reverse transcriptase. The
amplification efficiency is extremely high (10x
higher than PCR). LAMP is less prone to
inhibitors By designing 4 primers to
recognize 6 distinct regions, the LAMP method is
able to specifically amplify the target gene.
The total cost can be reduced, as LAMP does
not require special reagents or sophisticated
equipments. The amplified products have a
structure consisting of alternately inverted
repeats of the target sequence on the same
strand.
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Monitoring of LAMP products
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Quantitative analysis by real-time turbidimetry
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ABI microfluidic plate with prespotted
primers - 8 samples can be tested for 48
different targets. - Does not require liquid
handling robotics - Provides easy standardization
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LAMP product monitoring (summary)
(By-)products of LAMP (pyro phosphate) can be
used for target detection and quantification
Multiplex LAMP can be performed in
microwells and different micro- fluidic devices

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Confirmation and Monitoring

• DNA/RNA extractions on different substrates to be
  performed in the field and/or on-site
• White flies viruses from traps
• Bacterial pathogens from plant material
• Potato pathogens from tuber and leaves
• Monitoring
• Isothermal multiplex and simplex amplification
• LAMP (Optisense)
• Confirmation
• Isothermal multiplex and simplex amplification
• LAMP (Optisense)
• PLP in combination with RCA (Qlinea)

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First line screening (monitoring via fast semi
specific method)
Second line screening (confirmation via target
specific detection methods)
Real positive? Confirmation with another
technology
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Target ligation with universal LAMP primers
Exonuclease cutting
LAMP detection

LAMP
LAMP amplicon cutting
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PLP based multiplex detection
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Mechanism for amplified single molecule detection
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Working mechanism for amplified single molecule
detection
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RCA based quantitative multiplex detection
Signal amplification and specific labeling
Target recognition and circle creation
Microfluidic analysis
Thresholding
Sample
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• LAMP product confirmation
• Ligation based spec-LAMP (LS-LAMP) will
  probably support point mutation specific
  discrimination of targets in different array
  formats
• Ligation based Univ-LAMP (LU-LAMP) will be
  tested
• PLP RCA based confirmation
• High level of specificity and multiplexing
• Target recognition and amplification
  independent
• Universal downstream processing after ligation
• Ligation Fluorescently stained blobs can
  easily be discriminated and identified in a
  micro-fluidic device
• NAIMA confirmation
• Improvement of NAIMA universal amplification
  primers
• Hexaplex NAIMA amplification confirmed
• Microarray platform detection experimented on
  hexaplex NAIMA

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Deliverables D 7.1 Three protocols for DNA
extraction for selected set of targets in the
different matrices (Month 12) D 7.2 Provide
information to WP2 (partner 9) on the cost, time,
skill and conditions required for the
application of the detection method by
inspectors. And preliminary values for
parameters (or estimates) for uncertainties,
sampling, quality (sensitivity/specificity) and
protocols (Month 12). D 7.3 Two protocols for
isothermal amplification of selected targets
(Month 24) D 7.4 Three protocols for
single-plex/multiplex analysis of selected
targets (Month 30) D 7.5 Provide information to
WP2 (partner 9) on the uncertainties, sampling,
quality (sensitivity/specificity) and a full
protocol required for the detection method
developed (Month 30). D 7.6 Two Devices to be
used on-site (Month 30) D 7.7 Two monitoring
protocols (Month 36)
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• Task 7.1 DNA extractions (Month 12)
• Three novel extraction approaches (devices,
  set-ups, reagents) will be developed.
• Robustness of the three most promising
  extraction procedures will be determined.
• Applicability of three different extraction
  procedures for the full-range of material
  encountered by end-user inspectors (fruit tree
  material flowers, shoots, rootstocks,
  branches, fruit potato leaf, wood shavings (for
  PWN), vector insects (e.g. whitefly and
  Monochamus beetles) and isolated nematodes (e.g.
  trapped PWN). will be tested).
• Select and validate three on-site DNA
  extraction procedures to optimize assay
  sensitivity. Modification of three proven DNA
  extraction methods towards simplest possible
  manipulation for direct-in field application
  (e.g., one-step sample/DNA extraction in the
  field). This part has potential application for
  all project organisms.
• The three best candidate methods will be
  evaluated in the different laboratories.

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Task 7.1 DNA extractions (continued)
The following pathosystems will be addressed in
this WP PRI Targets pests will be white flies
(Bemisia tabaci) from traps ACW Target pests
will be bacterial fruit-tree pathogens (Erwinia
amylovora pome fruit Xanthmonas arboricola
pv. pruni stone fruit). NIB Target pests for
testing DNA extraction will be the potato brown
rot (Ralstonia solanacearum) and the potato ring
rot (Clavibacter michiganensis ssp. sepedonicus)
on potato leaves and tubers. Fera Targets will
be PWN directly on trapped nematodes, within
vector beetles and wood shaving material and
potato leaves. UNIBO Will participate on the
development of the extraction protocols for all
the bacterial species considered. In addition,
UNIBO will also focus on Pseudomonas syringae pv
actinidiae, an emerging pathogen that is
jeopardizing kiwifruit production in south
Europe. A new type of extraction and
amplification of target will be performed. CIP
Focus on DNA/RNA extraction of PYVV form
infected potato leaves. CAIQ Focus on DNA/RNA
extraction of Cotton leaf curl virus, transmitted
by white flies and also on Citrus greening.
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• Task 7.2 Generation of protocols for isothermal,
  singleplex and multiplex amplification of
  targets (Month 24).
• Two different isothermal amplification
  strategies will be tested for on-site
  confirmation LAMP and NAIMA.
• Optimized single-plex reaction procedures will
  be established for three key target pests (B.
  tabaci, Erwinia amylovora, Potato yellow vein
  virus) to enable assessing the performance of
  multiplex methods and to test limits of
  single-plex PCR methods with respect to speed
  and sensitivity.

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• Task 7.2 Generation of protocols for isothermal,
  single-plex and multiplex amplification of
  targets (Month 24).
• Multiplex reaction procedures using diverse
  technologies will be developed.
• The padlock probes technology will be
  investigated for simultaneous on-site detection
  of viruses (e.g. TYLCV, CYSDV, PYVV, TICV, ToCV,
  begomoviruses and including cotton leaf curl
  virus) within white flies (Bemisia tabaci).
• The combination of multiplex NAIMA amplification
  with easy-to-use portable detection systems will
  be developed for simultaneous detection of DNA
  and RNA pathogens. The target candidates will be
  the bacteria Ralstonia solanacearum and
  Clavibacter michiganensis ssp. sepedonicus, and
  one viral pathogen to be determined (Potato
  spindle tuber viroid (PSTVd), the Potato Virus Y
  (PVY) or the Pepino Mosaic Virus (PepMV)).
• The main focus will be placed on the robustness
  of the amplification process under variable
  conditions. Enzymes, buffers and amplification
  conditions (temperature, hold times, reagents
  etc) will be tested.
• PRI, Fera, ACW and NIB will collaborate in the
  testing and evaluation process.

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• Task 7.3 Generation of devices for multiplex
  detection on-site (Month 24)
• Qlinea will develop a prototype of a multiplex
  (10-20) single molecule detection device to be
  used for on-site application
• Optisense will provide access to a portable
  isothermal amplification reader to WP6 partners
  for validation purposes.
• PRI and Qlinea will collaborate in testing and
  evaluating the device developed by Qlinea for
  Bemisia transmitted virus detection.
• Fera and Optisense will collaborate in testing
  and evaluating the devices developed by Optisense
  for PWN and PYVV detection

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