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NIB activities in Q- DETECT WP7

NIB activities in Q- DETECT WP7. Vienna, 12 th June 2012 Dany Morisset , Rok Lenarčič, Tanja Dreo, Nataša Mehle, Maja Ravnikar. Targets: RNA : PSTV d ( Potato s pindle t uber v iroid ) DNA: brown rot ( Ralstonia solanacearum )

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NIB activities in Q- DETECT WP7

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  1. NIB activities in Q-DETECTWP7 Vienna, 12th June 2012 Dany Morisset, Rok Lenarčič, Tanja Dreo, Nataša Mehle, Maja Ravnikar

  2. Targets: • RNA : PSTVd (Potato spindle tuber viroid) • DNA: brown rot (Ralstonia solanacearum) • DNA: ring rot (Clavibacter michiganensis subsp. sepedonicus (colaboration with FERA, UK) PSTVd infection causes reduced tuber size and yield (left) when compared to non-infected tubers (EPPO, 2011). Ralstonia solanacearum infection causes brown rot in potato tubers (Pests and Diseases Image Library, 2011

  3. RT-LAMP for PSTVd detection Comparison of primer positions Real time RT-PCR for PSTVd (Boonham et al, 2004) R probe F RT-LAMP on PSTVd Tsutsumi et al., 2010 NIB RT-LAMP F2 LoopF F3 F2 LoopF F1 B1 LoopB B2 F3 B3 B3 B2 LoopB B1 F1

  4. RT-LAMP on PSTVd • Paper to be submitted • Single tube RT-LAMP: reverse transcription and LAMP reactions done in a single tube. • Results in usually 13-18 minutes, less than 25 minutes • (Tsutsumi RT-LAMP 35 mins with Optigene master mix) • Good sensitivity Dilution series of total plant RNA extraction

  5. RT-LAMP on PSTVd

  6. RT-LAMP on PSTVd PSTVd amplicon: specific melting temperature for confirmation of positive resultsvs. false positive. Box-plot of the melting temperatures of NIB-RT-LAMP products on viroid isolates. Melting temperatures were measured on the SmartCycler apparatus (Cepheid, Sunnyvale, CA)

  7. LAMP on Ralstonia solanacearum • Target genes: • 16S rRNA: good sensitivity and specificity, but shows cross reactivity with some potato extracts. Slow (up to 60min). Tm: 89.3°C +/- 0.5 °C • fliC (Kubota et al., 2008, 5 primers) improved with additional loop primer: speeds up reaction for ~7 minutes: results in max 20 minutes. Tm: 89.3°C +/- 0.2 °C and 92.2°C +/- 0.1 °C • egl: in silico covers all RS isolates, work in progress. Results in 13-17 min. Tm: 94.0°C +/- 0.2 °C • DNA extraction: • boiling of bacterial suspension in buffer/water Bacteria in water/buffer (106/ml), suspension boiled at 95°C for 2’ 5’ 10’ 15’ 20’ 30’ and tested by RS LAMP on 16S rRNA

  8. Specificity of Rs LAMP Additional 60 Rs isolates prepared to be tested for specificity studies

  9. Specificity of 16S Rs LAMP

  10. LAMP on Ralstonia solanacearum • egl gene: • Specific for R.solanacearum • 3 primer sets designed • very comparable performance • the best one chosen for further work (speed, strain coverage) • Preliminary data promising • Results in 15 mins, comparable to improved fliC LAMP • Sensitivity and specificity testing is in progress • Tested negative extracts showed no cross reactivity

  11. Multiplex LAMP – Rs and PSTVd Multiplexing R.solanacearum and PSTVd (reaction mix contains primer mix for RS fliCand PSTVd) Red triplicates have Tm and Tp specific for Rs (PSTVd cDNA is too diluted to be detected), blue triplicates have Tm and Tp specific for PSTVd cDNA (different for RNA) →The target that is in an exces gives signal

  12. Multiplex LAMP – other viroids V2: TCDVdV3: CEVdV4: CLVdV6: TASVdV7: CSVd →The signal is always Rs, the presence of other Qpest targets does not influence RS detection

  13. (reaction mix contains primer mix for RS fliC,PSTVd and COX) Close up of melting curves

  14. UNIVERSAL LAMP CONFIDENTIAL

  15. Future work • UNI-LAMP • CONFIDENTIAL • On-site DNA/RNA extraction • Optimise boiling • LFDs • Finish LAMP onegl • Optimisation/validation • Sensitivity • Finish specificity • Multiplex PSTVd-Rs • Optimise RNA/DNA co-detection with chosen Rs assay

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