Feb 18 th , 2013

1. On-site Confirmation and Monitoring Feb 18th, 2013 Cor Schoen

2. WP7. Confirmation and Monitoring • Main objective: To develop rapid, simple and reliable confirmatory and monitoring methods based on the detection of DNA

3. Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site White flies viruses from traps • Vector of Q-viruses: • Bemisia tabaci • Trialeurodes vaporariorum

4. Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site White flies viruses from traps Whitefly vectored viruses Crini viruses (ssRNA): Tomato chlorosis virus, (TOCV) Tomato infectious chlorosis virus, (TICV) Cucurbit yellow stunting disorder virus, (CYSDV) Potato yellow vein virus, (PYVV) Begomovirus (ssDNA): Tomato yellow leaf curl virus, (TYLCV) Cotton leaf curl virus (CLCuV)

5. Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site • White flies viruses from traps DNA/RNA Extraction Criteria: - Easy - Fast - Efficient - Cheap

6. Extraction procedure (Epicentre) Place white fly in tube Homogenize Whitefly material by pottering • Add 20 ul of QuickExtract RNA/DNA extraction buffer solution into an eppendorf tube • Add one or more white flies to this tube • Homogenize the white flies by pottering (30 sec) • Take out 1 ul extract and add to LAMP reagent Incubate the tube at 65oC for 10-20 min and measure fluorescence Pottering of the white fly

7. Amplification To enable on-site detection, (isothermal) methods in one reaction mixture were tested  - PCR - PLP - RCA - NAIMA - LAMP    We selected: • LAMP (loop –mediated isothermal amplification)

8. LAMP characteristics • There is no need for a step to denature double stranded DNA into a single stranded form • The whole amplification reaction takes place continuously under isothermal conditions • Amplification can be done with RNA templates following the same procedure as with DNA templates, simply through the addition of reverse transcriptase • The amplification efficiency is extremely high • LAMP is less prone to inhibitors • The total cost can be reduced, as LAMP does not require special reagents or sophisticated equipment

9. Monitoring of LAMP products Genie II Amplification plot Dissociation plot

10. LAMP development Developed LAMP detection methods in whitefly for use by national plant protection organizations (NPPO) and inspection services Whitefly vectored viruses Crini viruses (ssRNA): Tomato chlorosis virus, (TOCV) Tomato infectious chlorosis virus, (TICV) Cucurbit yellow stunting disorder virus, (CYSDV) Potato yellow vein virus, (PYVV) Begomovirus (ssDNA): Tomato yellow leaf curl virus, (TYLCV) Cotton leaf curl virus (CLCuV)

11. LAMP validation Tovalidate LAMP on different whiteflies, TaqManassayswereused as a control:

12. LAMP TYLCV on white flies compared to TaqMan • All TYLCV (ssDNA) infected white flies (1 or 5) positively tested with TaqMan, can be detected with LAMP

13. Monitoring (first/second line screening) Extraction DNA / RNA Reagent First line screening (monitoring via fast semi specific method, 15-20 min) - + Second line screening (confirmation via target specific detection methods, 2-3 hours) Χ Specific target detection

14. Monitoring (first line screening) Clondiag Luminex Combination of TICV, ToCV and TYLCV in LAMP

15. MultiplexTYLCV, TOCV and TICV Monitoring(second line screening) Clondiag Tech Clondiag Luminex Luminex Technologie

16. Can we set up a specific universal multiplex LAMP? • Criteria: • Easy design • Isothermal multiplex amplification • Fast • Sensitive • Applicable for DNA

17. Ligation based Universal-LAMP Ligation based Universal-LAMP Target ligation with universal LAMP primers LAMP Amplification Luminex bead Luminex LAMP detection LAMP Amplicon ClonDiag detection

18. Ligation based Universal-LAMP • Advantages: • Development of specific sites are easy; small discriminatory areas can be used • Point mutation discrimination • Universal LAMP primers (inexpensive) • Multiplexing of targets is simple • Possible disadvantages: • More steps involved; before starting of the LAMP, ligation of the LAMP probes have to be performed on target DNA

19. Ligation based Universal-LAMP (workflow) DNA extraction Target ligation with 2 unique linear probe pairs LAMP sample mix Multiplex detection

20. Ligation based Universal-LAMP • Different DNA targets can be independently detected in one LAMP reaction • Point mutation discrimination has not yet been realised

21. C • Biotin-LAMP • LAMP target ( RNA/DNA) amplification with biotinylated nucleotides allows on-site multiplex detection with Luminex or ClonDiag • Ligation based Universal-LAMP • Different DNA targets can be independently detected in one LAMP reaction • Limit of detection is 100fg for each target • Point mutation discrimination has not yet been realised Monitoring(second line screening)

22. Perspectives

23. Combined amplification and detection To simplify the procedure, amplification and detection can be combined in a single system: Microfluidics Microfluidics allow multiple reactions combined with detection (PRI)

24. Acknowledgements • Odette Mendes • Cor Schoen Plant Research International B.V. Wageningen, The Netherlands Food and Environment Research Agency (Fera), United Kingdom - Jenny Tomlinson - Niek Kunst Radboud University Nijmegen, Institute for Molecules and Materials, The Netherlands • Duncan Clark • Michael Andreou Optigene Ltd. West Sussex, England