Transcript WP7_PRI - q
On-site Confirmation and Monitoring Cor Schoen Feb 18th, 2013 WP7. Confirmation and Monitoring Main objective: To develop rapid, simple and reliable confirmatory and monitoring methods based on the detection of DNA 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 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) 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 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 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) 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 Monitoring of LAMP products Genie II Amplification plot Dissociation plot 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) LAMP validation To validate LAMP on different white flies, TaqMan assays were used as a control: LAMP TYLCV on white flies compared to TaqMan TaqMan Sample ↓ 1 WF 1 TYLCV 1 WF 2 TYLCV 1 WF 3 TYLCV Lf TYLCV-x Lf TYLCV 3181291 1 WF Bt 1 1 WF Bt 2 1 WF Bt 3 Lf 1 11-01 Lf 2 11-01 Lf 3 11-01 Lf + 11-01 5 WF tube 3 15-7 1 WF tube 3 15-7 5 WF tube 4 15-7 1 WF tube 4 15-7 5 WF tube 8 15-7 1 WF tube 8 15-7 5 WF tube 9 15-7 1 WF tube 9 15-7 5 WF tube 3 19-7 1 WF tube 3 19-7 5 WF tube 4 19-7 1 WF tube 4 19-7 TYLCV Ct +/- IL +/- SAR 30,45 + 27,66 + 27,31 + 15,50 + 29,67 undet undet 19,73 25,90 29.8 25.58 undet 27.96 24.07 29.89 39.54 30.73 undet 28.09 31.32 + + + + + + + + + + + + 34.5 - LAMP TYLCV TYLCV 1380 1380 14-mrt 24-mrt + + + + + + + + + + + + + + + + + + TaqMan Sample ↓ 5 WF tube 9 19-7 1 WF tube 1 1 WF tube 2 1 WF tube 3 1 WF tube 4 1 WF tube 5 1 WF tube 7 1 WF tube 8 1 WF tube 9 1 WF large tube 5 WF tube 4 5 WF tube 8 5 WF tube 9 1 WF tube 2 Amir 1 WF tube 3 Amir 1 WF tube 3.1 1 WF tube 3.2 1 WF tube 3.3 1 WF tube 8.1 1 WF tube 8.2 1 WF tube 8.3 1 WF tube 9.1 1 WF tube 9.2 1 WF tube 9.3 Ct 28.25 30.25 undet undet undet 34.57 undet 32.68 33.04 39.44 35.9 26.93 28.54 undet 32.05 35.18 38.07 37.9 undet undet undet undet undet undet TYLCV +/- IL +/- SAR + + + + + 37.11 + + + + + + + + - LAMP TYLCV TYLCV 1380 1380 14-mrt 24-mrt + + + + + + + + + + + + + - All TYLCV (ssDNA) infected white flies (1 or 5) positively tested with TaqMan, can be detected with LAMP 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 Monitoring (first line screening) Clondiag Luminex Combination of TICV, ToCV and TYLCV in LAMP Monitoring (second line screening) Clondiag Tech Clondiag Luminex Luminex Technologie Multiplex TYLCV, TOCV and TICV Can we set up a specific universal multiplex LAMP? Criteria: Easy design Isothermal multiplex amplification Fast Sensitive Applicable for DNA Ligation based Universal-LAMP Ligation based Universal-LAMP Target ligation with universal LAMP primers Luminex bead LAMP Amplification Luminex LAMP detection LAMP Amplicon ClonDiag detection 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 Ligation based Universal-LAMP (workflow) DNA extraction Target ligation with 2 unique linear probe pairs LAMP sample mix Multiplex detection Ligation based Universal-LAMP Different DNA targets can be independently detected in one LAMP reaction Point mutation discrimination has not yet been realised C Monitoring (second line screening) 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 Perspectives 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) Acknowledgements Plant Research International B.V. Wageningen, The Netherlands - Odette Mendes - Cor Schoen Food and Environment Research Agency (Fera), United Kingdom - Jenny Tomlinson Radboud University Nijmegen, Institute for Molecules and Materials, The Netherlands - Niek Kunst Optigene Ltd. West Sussex, England - Duncan Clark - Michael Andreou