On-site Confirmation and Monitoring
Cor Schoen
Feb 18th, 2013
WP7. Confirmation and Monitoring

Main objective: To develop rapid, simple and reliable
confirmatory and monitoring methods based on the
detection of DNA
Confirmation and Monitoring
 DNA/RNA extractions on different substrates to be
performed in the field and/or on-site
White flies viruses from traps
Vector of Q-viruses:
 Bemisia tabaci
 Trialeurodes vaporariorum
Confirmation and Monitoring
 DNA/RNA extractions on different substrates to be
performed in the field and/or on-site
White flies viruses from traps
Whitefly vectored viruses
Crini viruses (ssRNA):
Tomato chlorosis virus, (TOCV)
Tomato infectious chlorosis virus, (TICV)
Cucurbit yellow stunting disorder virus, (CYSDV)
Potato yellow vein virus, (PYVV)
Begomovirus (ssDNA):
Tomato yellow leaf curl virus, (TYLCV)
Cotton leaf curl virus (CLCuV)
Confirmation and Monitoring
 DNA/RNA extractions on different substrates to be
performed in the field and/or on-site
White flies viruses from traps
DNA/RNA Extraction Criteria:
-
Easy
Fast
Efficient
Cheap
Extraction procedure (Epicentre)
Place white fly in tube
Homogenize Whitefly material by pottering
 Add 20 ul of QuickExtract RNA/DNA extraction
buffer solution into an eppendorf tube
 Add one or more white flies to this tube
 Homogenize the white flies by pottering (30 sec)
 Take out 1 ul extract and add to LAMP reagent
Incubate the tube at 65oC for 10-20 min and
measure fluorescence
Pottering of the white fly
Amplification
To enable on-site detection, (isothermal) methods in one
reaction mixture were tested
- PCR
- PLP - RCA
- NAIMA
- LAMP




We selected:
LAMP (loop –mediated isothermal amplification)
LAMP characteristics
 There is no need for a step to denature double stranded DNA
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
 LAMP is less prone to inhibitors
 The total cost can be reduced, as LAMP does not require
special reagents or sophisticated equipment
Monitoring of LAMP products
Genie II
Amplification plot
Dissociation plot
LAMP development
Developed LAMP detection methods in whitefly for use by
national plant protection organizations (NPPO) and inspection
services
Whitefly vectored viruses
Crini viruses (ssRNA):
Tomato chlorosis virus, (TOCV)
Tomato infectious chlorosis virus, (TICV)
Cucurbit yellow stunting disorder virus, (CYSDV)
Potato yellow vein virus, (PYVV)
Begomovirus (ssDNA):
Tomato yellow leaf curl virus, (TYLCV)
Cotton leaf curl virus (CLCuV)
LAMP validation
To validate LAMP on different white flies, TaqMan
assays were used as a control:
LAMP TYLCV on white flies compared to TaqMan
TaqMan
Sample ↓
1 WF 1 TYLCV
1 WF 2 TYLCV
1 WF 3 TYLCV
Lf TYLCV-x
Lf TYLCV 3181291
1 WF Bt 1
1 WF Bt 2
1 WF Bt 3
Lf 1 11-01
Lf 2 11-01
Lf 3 11-01
Lf + 11-01
5 WF tube 3 15-7
1 WF tube 3 15-7
5 WF tube 4 15-7
1 WF tube 4 15-7
5 WF tube 8 15-7
1 WF tube 8 15-7
5 WF tube 9 15-7
1 WF tube 9 15-7
5 WF tube 3 19-7
1 WF tube 3 19-7
5 WF tube 4 19-7
1 WF tube 4 19-7
TYLCV
Ct
+/- IL +/- SAR
30,45
+
27,66
+
27,31
+
15,50
+
29,67
undet
undet
19,73
25,90
29.8
25.58
undet
27.96
24.07
29.89
39.54
30.73
undet
28.09
31.32
+
+
+
+
+
+
+
+
+
+
+
+
34.5
-
LAMP
TYLCV
TYLCV
1380
1380
14-mrt
24-mrt
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
TaqMan
Sample ↓
5 WF tube 9 19-7
1 WF tube 1
1 WF tube 2
1 WF tube 3
1 WF tube 4
1 WF tube 5
1 WF tube 7
1 WF tube 8
1 WF tube 9
1 WF large tube
5 WF tube 4
5 WF tube 8
5 WF tube 9
1 WF tube 2 Amir
1 WF tube 3 Amir
1 WF tube 3.1
1 WF tube 3.2
1 WF tube 3.3
1 WF tube 8.1
1 WF tube 8.2
1 WF tube 8.3
1 WF tube 9.1
1 WF tube 9.2
1 WF tube 9.3
Ct
28.25
30.25
undet
undet
undet
34.57
undet
32.68
33.04
39.44
35.9
26.93
28.54
undet
32.05
35.18
38.07
37.9
undet
undet
undet
undet
undet
undet
TYLCV
+/- IL +/- SAR
+
+
+
+
+
37.11
+
+
+
+
+
+
+
+
-
LAMP
TYLCV
TYLCV
1380
1380
14-mrt
24-mrt
+
+
+
+
+
+
+
+
+
+
+
+
+
-
 All TYLCV (ssDNA) infected white flies (1 or 5) positively tested with TaqMan,
can be detected with LAMP
Monitoring (first/second line screening)
Extraction
DNA / RNA
Reagent
First line screening
(monitoring via fast semi
specific method, 15-20 min)
+
Second line screening
(confirmation via target specific
detection methods, 2-3 hours)
Χ
Specific target detection
Monitoring (first line screening)
Clondiag
Luminex
Combination of TICV, ToCV and TYLCV in LAMP
Monitoring (second line screening)
Clondiag Tech
Clondiag
Luminex
Luminex Technologie
Multiplex
TYLCV, TOCV and TICV
Can we set up a specific universal multiplex LAMP?
Criteria:
 Easy design
 Isothermal multiplex amplification
 Fast
 Sensitive
 Applicable for DNA
Ligation based Universal-LAMP
Ligation based Universal-LAMP
Target ligation with
universal LAMP primers
Luminex
bead
LAMP
Amplification
Luminex LAMP detection
LAMP
Amplicon
ClonDiag detection
Ligation based Universal-LAMP
Advantages:
- Development of specific sites are easy; small
discriminatory areas can be used
- Point mutation discrimination
- Universal LAMP primers (inexpensive)
- Multiplexing of targets is simple
Possible disadvantages:
- More steps involved; before starting of the LAMP, ligation
of the LAMP probes have to be performed on target DNA
Ligation based Universal-LAMP (workflow)
DNA extraction
Target ligation with
2 unique linear probe pairs
LAMP sample mix
Multiplex detection
Ligation based Universal-LAMP
 Different DNA targets can be independently detected in
one LAMP reaction
 Point mutation discrimination has not yet been realised
C Monitoring (second line screening)
Biotin-LAMP
 LAMP target ( RNA/DNA) amplification with biotinylated
nucleotides allows on-site multiplex detection with Luminex
or ClonDiag
Ligation based Universal-LAMP
 Different DNA targets can be independently detected in one
LAMP reaction
 Limit of detection is 100fg for each target
 Point mutation discrimination has not yet been realised
Perspectives
Combined amplification and detection
To simplify the procedure, amplification and detection can be
combined in a single system:
Microfluidics
Microfluidics allow multiple reactions combined with detection (PRI)
Acknowledgements
Plant Research International B.V.
Wageningen, The Netherlands
- Odette Mendes
- Cor Schoen
Food and Environment Research
Agency (Fera), United Kingdom
- Jenny Tomlinson
Radboud University Nijmegen,
Institute for Molecules and
Materials, The Netherlands
- Niek Kunst
Optigene Ltd.
West Sussex, England
- Duncan Clark
- Michael Andreou