WP7, partner 1 February 2013 Jenny Tomlinson, Neil Boonham.

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Presentation transcript:

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

Universal LAMP: arrays Ligation – LAMP with biotin dCTP – hybridisation - detection

LAMP for potato yellow vein virus PYVV assayplant 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 B3 3’ 5’ 3’ F1c F2 B2 B1c 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 min min min stem primers can increase the speed of amplification

LAMP for Guignardia citricarpa Wellstrip 1strip 2 (-30C then 4C)strip 3 (-30C)strip 4 (4C)strip 5 (ice then 4C)strip 6 (ice then -30C) : : : : : : : : : : : : : : : : : : : : : : : Wellstrip 1strip 2 (-30C then 4C)strip 3 (-30C)strip 4 (4C)strip 5 (ice then 4C)strip 6 (ice then -30C) : : : (+) (+) : : : : G. citricarpa L. huidobrensis Complete master mix (ie containing primers) is not stable at 4°C

Reagent stability Wellday 6day 8day 16day : : : : : : : : 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) TaqMan +- LAMP 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 Run LAMP on Genie II instrument 35 minutes up to 14 samples Approx. 30 minutes hands-on time to test 14 samples (mostly sampling)

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 negative control positive control sample 1sample 2sample 3sample 4sample 5sample 6 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