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