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WP7, partner 1 February 2013

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.

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WP7, partner 1 February 2013

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

  2. LAMP-based detection of plant pests and pathogens: partner 1 • Universal-LAMP for multiplex detection • LAMP assays for individual targets (viruses, fungi, insects)

  3. 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

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

  5. LAMP for potato yellow vein virus PYVV 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

  6. 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

  7. 5’ 3’ 3’ 5’ 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 F2 F3c F2c FL F1c B1 BLc B2 B3 F3 F2 FLc F1 B1c BL B2c B3c B2 B1c

  8. LAMP for quarantine Liriomyza • loop • stem • loop • + stem 23-24 min stem primers can increase the speed of amplification • + loop • stem + loop + stem 17-18 min 12-13 min

  9. LAMP for Guignardia citricarpa G. citricarpa L. huidobrensis Complete master mix (ie containing primers) is not stable at 4°C

  10. Reagent stability G. citricarpa: reagents stored at approx 4°C as separate primer mix and master mix

  11. 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% positive predictive value = 96% negative predictive value = 95% sensitivity = 90% specificity = 98%

  12. LAMP for Chalara fraxinea Take sample from leading edge of lesion 2-5 minutes per sample Approx. 30 minutes hands-on time to test 14 samples (mostly sampling) Place in tube with PEG buffer and shake for 1 minute 1 minute up to 8 samples Transfer approx. 10 µl into tube containing 90 µl water using innoculating loop if in the field 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

  13. LAMP for detection of Chalara fraxinea in wood samples 1. Preparation of samples LAMP can be incorporated into simple field methods Block A: C. fraxinea strip Block B: COX strip Shake for 1 minute Dilute 1 in 10 in water (use large loop) 1 2 3 4 5 6 7 8 2. Preparation of test strips negative control sample 1 sample 2 sample 3 sample 4 sample 5 sample 6 positive control C. fraxinea strip COX strip Transfer to strip (use small loop) Test up to six samples at once

  14. Acknowledgements • Sioban Ostoja-Starzewska • Catherine Harrison • Ian Dawson • NIB and PRI • Optigene • ACW and University of Padova

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