WP7, partner 1 February 2013 Jenny Tomlinson, Neil Boonham.
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Transcript of WP7, partner 1 February 2013 Jenny Tomlinson, Neil Boonham.
WP7, partner 1February 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
Universal LAMP: arrays
Ligation – LAMP with biotin dCTP – hybridisation - detection
LAMP for potato yellow vein virusPYVV assay plant assay
Sensitivity was improved (10-fold) by use of a 2-
temperature 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...
F3c F2c
B2c B3cF3 F2
B2 B33’5’
5’3’
F1cF2
B2B1c
F1c
F1
B1
B1c
FL
FLc
BLc
BL
stem primers: can be in either orientation
LAMP for quarantine Liriomyza- loop- stem
- loop+ stem
+ loop- stem
+ loop+ stem
23-24 min
12-13 min17-18 min
stem primers can increase the speed
of amplification
LAMP for Guignardia 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 84.49
6 19:22 86.99 23:12 86.84 21:27 86.69 21:48 86.94 19:03 84.16 20:40 86.89
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 - - - - - - - - - - - 82.242 - - - - - - 21:06 82.96 - - 27:21 81.963 20:33 79.79 (+) 79.43 - - (+) 79.41 - 81.55 25:36 80.494 27:03 79.54 - 82.49 - - 24:36 82.01 26:06 81.49 - -
G. citricarpa
L. huidobrensis
Complete master mix (ie containing primers) is not stable at 4°C
Reagent stability
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
G. citricarpa: reagents stored at approx 4°C as separate primer mix and master mix
LAMP for Chalara fraxinea
• Rapid extraction method for wood• Alkaline PEG buffer, manual shaking and dilution• Laboratory validation (150 samples)• Trial field deployment (ongoing)
positive predictive value = 96%
negative predictive value = 95%
sensitivity = 90%
specificity = 98%
prevalence = 34%
LAMP for Chalara fraxinea
Take sample from leading edge of lesion
Transfer approx. 1 µl per LAMP reaction
Place in tube with PEG buffer and shake for 1 minute
Transfer approx. 10 µl into tube containing 90 µl water
2-5 minutes per sample
using innoculating loop if in the field
1 minute up to 8 samples
2 minutes up to 14 samples
2 minutes up to 14 samples
Run LAMP on Genie II instrument 35 minutes up to 14 samples
Approx. 30 minutes hands-on time to test 14 samples (mostly
sampling)
1 2 3 4 5 6 7 8
Shake for 1 minute Dilute 1 in 10 in water
(use large loop)
Block A:C. fraxinea
strip
Block B:COX strip
1. Preparation of samples
2. Preparation of test strips
C. fraxinea strip
COX strip
LAMP for detection of Chalara fraxinea in wood samples
Transfer to strip(use small loop) Test up to six samples at once
LAMP can be incorporated into
simple field methods
Acknowledgements
• Sioban Ostoja-Starzewska• Catherine Harrison• Ian Dawson• NIB and PRI• Optigene• ACW and University of Padova