On-Site Confirmation and Monitoring
Cor Schoen
29th Nov, 2010 Q-detect
WP7. On-Site Confirmation and Monitoring

Main objective: To develop rapid, simple and
reliable confirmatory and monitoring methods
based on the detection of DNA
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
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
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
Boonham et al 2008
Boonham et al 2008
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
To validate LAMP, NAIMA and PLP-RCA, different
TaqMan assays were developed as the golden
standard for target detection:
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)
Extraction
DNA / RNA
React.
Mix
First line screening
(monitoring via fast
semi specific method)
+
Χ
Second line screening
(confirmation via target
specific detection methods)
Specific target detection
Real positive?
Confirmation with another
technology
Monitoring (first line screening)
Isothermal multiplex and simplex amplification
• Easy
• Fast
• Sensitive
• Applicable for DNA and RNA
- PCR
- PLP - RCA
- NAIMA
- LAMP




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
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.
Monitoring of LAMP products
Quantitative analysis by real-time turbidimetry
Quantitative analysis by real-time turbidimetry
(A) 1:2×109copies.
5
5:2×10 copies.
8
2:2×10 copies.
4
6:2×10 copies.
7
3:2×10 copies.
3
7:2×10 copies.
6
4:2×10 copies.
8:without template DNA
(B) The relation between the threshold times (Tt) of each sample and log of the amount of initial
template DNA (mean ±S.D. : n=3)
Multiplex LAMP product measurement with micro-wells or micro-fluidics
ABI microfluidic plate with prespotted primers:
- 8 samples can be tested for 48 different targets.
- Does not require liquid handling robotics
- Provides easy standardization
Detection of LAMP products
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 microfluidic devices
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)
First line screening
(monitoring via fast
semi specific method)
Second line screening
(confirmation via target
specific detection methods)
Real positive?
Confirmation with another technology
Ligation based Univ-LAMP (LU-LAMP) for point mutation specific multiplexing
Target ligation
Biotin/Strep binding
LAMP detection
LAMP detection
LAMP ampl/cutting
Capturing
LAMP ampl/cutting
Washing/ release
Ligation based Univ-LAMP (LU-LAMP) for point mutation specific multiplexing
Target ligation with
universal LAMP primers
Exonuclease cutting
LAMP detection
+
LAMP amplicon
cutting
LAMP
PLP based multiplex detection
Mechanism for amplified single molecule detection
Working mechanism for amplified single molecule detection
RCA based quantitative multiplex detection
Target recognition
and circle creation
Signal amplification
and specific labeling
Microfluidic analysis
Sample
Thresholding
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
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)
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.
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
shaving material and potato leaves.
wood
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.
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.
Task 7.2 Generation of protocols for isothermal, singleplex 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.
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