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NIB activities in Workpackage 7

NIB activities in Workpackage 7. Rok Lenarčič, Dany Morisset, Maja Ravnikar February, 2013. NIB activities in Workpackage 7. PSTVd RT-LAMP Single tube reaction, 25’ to final answer Sensitivity 10x better than RT-PCR, 100x lower than RTqPCR Detects all tested PSTVd isolates,

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NIB activities in Workpackage 7

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  1. NIB activities in Workpackage 7 Rok Lenarčič, Dany Morisset, Maja Ravnikar February, 2013

  2. NIB activities in Workpackage 7 • PSTVd RT-LAMP • Single tube reaction, 25’ to final answer • Sensitivity 10x better than RT-PCR, 100x lower than RTqPCR • Detects all tested PSTVd isolates, • Paper accepted published in Plant Pathology journal • Ralstonia solanacearum LAMP • 3 LAMP sets evaluated (fliC, 16S rRNA, egl - 3 primer sets) • LAMP on endoglucanase gene was selected as the best • Detects all isolates from Phylotype I, II and III and most of Phylotype IV (negative proposed to form a new phylotype/species) • Total running time 30’ • Boiling without DNA extraction is sufficient before LAMP • Paper in preparation • Multiplexing • Simultaneous detection of RNA (PSTVd) and DNA (Rs) targets works in one tube. Work in progress • Ligation based Universal LAMP (done in collaboration with FERA, UK) • Ligation optimised • Problematic ‘Loop’ primers swaped for ‘Stem’ primers (Gandelman et al., 2011) • Exonuclease treatment tested (probably not worth regarding gain vs. time+cost) • Multiplexing on DNA targets works • Biotinylation works for detection on arrays

  3. PSTVd detection by RT-LAMP Paper published in Plant Pathology Reverse transcription combined with LAMP→ single tube reaction Run time 25 mins Faster and more efficient amplification than with previously reported RT-LAMP

  4. Specificity of the PSTVd RT-LAMP Detects all tested PSTVd and TPMVd and some TCDVd isolates (similar sequence, same origin) PSTVd has different Tm than TPMVd. 16 PSTVd isolates and 42 Pospiviroid isolates (TPMVd, TCDVd, TASVd, CSVd, CEVd, CLVd). One isolate from the genus Cocadviroid (Pospiviroidae), and five isolates from the family Avsunviroidae tested.

  5. Sensitivity of the PSTVd RT-LAMP • 10x more sensitive than RT-PCR • 100x less sensitive than real time RT-PCR

  6. Comparison of different LAMP assays for Ralstonia solanacearum detection

  7. R. solanacearum detection by LAMP • LAMP on 3 targets: • fliC (Kubota et al., 2008) additional loop primer, optimised primer mix • 16S rRNA. Based on Weller’s qPCR assay • egl (3 primer sets designed, one chosen for further validations) *depends on phylotype ** phylotype IIB

  8. R. solanacearum isolates *Phylotype IV including Blood Disease Bacterium and R. syzygii which were proposed to form a separated species (Remenant et al. 2011) ** 2 R. solanacearum detected + 2 R. syzygii, except from a genetically distant strain RUN14 that is proposed to form a new phylotype V *** Observed signal is not reproducible, and can be distinguished from real positive based on Tm

  9. Sensitivity of egl LAMP for R. solanacearum detection qPCR sensitivity marked with bold line adetected once out of three replicates bdetected twice out of three replicates

  10. egl LAMP Tm Phylotypes I and III: 94.6°C Phylotypes IIA, IIB, IV: 93.8°C

  11. Multiplex LAMP (DNA & RNA) Multiplexing R. solanacearum (DNA) and PSTVd (RNA). Dilutions made in dH2O (extracts might cause problems). Usually one product is detectable, rarely both (example below) Triplex testing in progress (R.solanacearum, C. michiganensis sepedonicus, PSTVd)

  12. Future work • Complete multiplexing story • sensitivity • specificity • Ligation universal LAMP: • To be discussed between developers?

  13. NAIMA • Initially proposed in Qdetect • NAsba Integrated Multiplex Amplification

  14. PSTVd • Starting material: RNA • Same region as for Boonham’s one step RT-PCR (2004) (used in routine lab) • Amplification up to 7.107 in 45min • About 60x less sensitive than qPCR • Specificity OK • Test done on several isolates (PSTVd and other viroids)

  15. Ralstonia solanacearum • Starting material: DNA • Targeting 16S RNA gene. • Very good amplification rates for all (108 to 109 in 45min). • Sensitivity comparable to that of qPCR (Weller). • Specificity OK • Ralstonia solanacearum strains from three phylotypes + BDB and R. syzygii (phyIV) tested and detected. • Cross reaction strains requested by EU regulation collected (other Ralstonia sp., Bacillus polymixa, Burkholderiaceae ...). All negative except R. mannitolilytica (also detected by qPCR) • No amplification in potato extract used as negative samples

  16. Duplex PSTVd-Ralstonia • Starting material: DNA, RNA or mixture of both • Duplex primers mixture • Sensitivity for a single target is same as in singleplex reactions • When both targets are present in different amounts, amplification is good but the target at low concentration is sometimes not amplified

  17. NAIMA products detection • Adding of biotinylated-UTP (up to 1/3) in the reaction does not affect the amplification • Clondiag Array Tubes for confirmation • 110 min from product hybridization to data analysis (to be improved) • Multiplexing is conserved • Array tube (14x14) just received • Test in progress

  18. Acknowledgements NIB: • Dany Morisset • Rok Lenarčič • Maja Ravnikar • Nataša Mehle • Tanja Dreo • Manca Pirc • David Dobnik (NAIMA) FERA: • Jenny Tomlinson • Neil Boonham • PRI: • Cor Schoen

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