WP7, partner 1
February 2013
Jenny Tomlinson, Neil Boonham
LAMP-based detection of plant
pests and pathogens: partner 1
• Universal-LAMP for multiplex detection
• LAMP assays for individual targets (viruses, fungi,
insects)
Universal LAMP: arrays
• Biotin-dUTP in LAMP (ISO mastermix – incorporation of dUTP...)
• Higher concentration – better signals on array
• Ligation probe containing ZIP code sequence; 0.125U Pfu; UNI LAMP, 140
µM biotin dUTP, F-stem rev only; hybridisation to ZIP code array
80000
30000
60000
20000
40000
10000
20000
0
0
-20000
0
10
20
30
40
75
30000
60000
20000
40000
80
85
90
95
-10000
80000
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
10000
20000
0
0
-20000
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
0
10
20
30
40
75
-10000
80
85
90
95
Universal LAMP: arrays
Ligation – LAMP with biotin dCTP – hybridisation - detection
LAMP for potato yellow vein virus
PYVV assay
plant assay
Sensitivity was improved
(10-fold) by use of a 2temperature protocol (initial
step 10 minutes at 50°C)
before incubation at 65°C
LAMP for quarantine Liriomyza
• L. huidobrensis, L. trifolii, L. sativae; Liriomyza spp. control
• Different life stages tested (pupae, empty pupal cases, eggs,
leaf mines)
1/100
dilution
1/10 dilution
LAMP for quarantine Liriomyza
• Stem primers (Gandelman et al 2011)
• To replace loop primers – see work with NIB on Uni LAMP
• To use with loop primers to increase speed of reaction...
stem primers: can be in
either orientation
F1c
3’
5’
F2
F3c F2c
FL
F1c
B1
BLc
B2 B3
F3 F2
FLc
F1
B1c
BL
B2c B3c
B2
B1c
5’
3’
LAMP for quarantine Liriomyza
- loop
- stem
- loop
+ stem
23-24 min
+ loop
- stem
stem primers can
increase the speed
of amplification
+ loop
+ stem
17-18 min
12-13 min
LAMP for Guignardia citricarpa
G. citricarpa
Well
strip 1
strip 2 (-30C then 4C)
strip 3 (-30C)
strip 4 (4C)
strip 5 (ice then 4C)
strip 6 (ice then -30C)
1
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
3
22:22
86.84
22:12
86.99
21:42
86.61
22:48
86.64
24:48
86.91
21:25
86.76
4
20:37
86.85
26:57
86.7
22:27
86.81
24:03
86.99
20:33
86.91
19:25
87.05
5
20:07
86.7
19:42
86.8
19:27
86.92
23:33
86.7
23:18
86.82
6
19:22
86.99
23:12
86.84
21:27
86.69
21:48
86.94
19:03
84.16
84.49
20:40
86.89
L. huidobrensis
Well
strip 1
strip 2 (-30C then 4C)
strip 3 (-30C)
strip 4 (4C)
strip 5 (ice then 4C)
strip 6 (ice then -30C)
1
-
-
-
-
-
-
-
-
-
-
-
82.24
2
-
-
-
-
-
-
21:06
82.96
-
-
27:21
81.96
3
20:33
79.79
(+)
79.43
-
-
(+)
79.41
-
81.55
25:36
80.49
4
27:03
79.54
-
82.49
-
-
24:36
82.01
26:06
81.49
-
-
Complete master mix (ie containing primers) is not stable at 4°C
Reagent stability
G. citricarpa: reagents stored at approx 4°C as separate primer
mix and master mix
Well
day 6
day 8
day 16
day 48
1
-
-
-
-
-
-
-
-
2
14:30
90.54
16:00
90.29
15:00
90.63
25:45
90.54
3
16:30
90.48
17:30
90.63
18:00
90.68
20:30
90.48
LAMP for Chalara fraxinea
•
•
•
•
Rapid extraction method for wood
Alkaline PEG buffer, manual shaking and dilution
Laboratory validation (150 samples)
Trial field deployment (ongoing)
prevalence = 34%
LAMP
TaqMan
+
-
+
46
2
positive predictive value = 96%
-
5
97
negative predictive value = 95%
sensitivity specificity
= 90%
= 98%
LAMP for Chalara fraxinea
Approx. 30 minutes hands-on
time to test 14 samples (mostly
sampling)
using innoculating
loop if in the field
Take sample from leading
edge of lesion
Place in tube with PEG buffer
and shake for 1 minute
2-5 minutes per sample
1 minute up to 8 samples
Transfer approx. 10 µl into
tube containing 90 µl water
2 minutes up to 14 samples
Transfer approx. 1 µl per
LAMP reaction
2 minutes up to 14 samples
Run LAMP on Genie II
instrument
35 minutes up to 14 samples
LAMP for detection of Chalara fraxinea in wood samples
1. Preparation of samples
LAMP can be
incorporated into
simple field
methods
Shake for 1 minute
Dilute 1 in 10 in
water
(use large loop)
negative control
sample 1
sample 2
sample 3
sample 4
sample 5
sample 6
positive control
2. Preparation of test strips
C. fraxinea
strip
1 2 3 4 5 6 7 8
COX
strip
Transfer to strip
(use small loop)
Test up to six samples at once
Block A: Block B:
C. fraxinea COX strip
strip
Acknowledgements
•
•
•
•
•
•
Sioban Ostoja-Starzewska
Catherine Harrison
Ian Dawson
NIB and PRI
Optigene
ACW and University of Padova