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SynTura: A New Ribonuclease Resistant Viral RNA Control Material

SynTura: A New Ribonuclease Resistant Viral RNA Control Material. Ralf Schönbrunner, Ph.D. SoGAT XXI Brussels, Belgium May 28, 2009. SynTura Project.

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SynTura: A New Ribonuclease Resistant Viral RNA Control Material

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  1. SynTura: A New Ribonuclease Resistant Viral RNA Control Material Ralf Schönbrunner, Ph.D. SoGAT XXI Brussels, Belgium May 28, 2009

  2. SynTura Project • The SynTura project was created to provide RNA Internal Controls and Quantification Standards which behave like the target they intend to control • Synthetic & Natural = SynTura

  3. SynTura Project Objectives • Develop technology platform which can provide control material close to real analyte • HCV was used as model system • Also functional as QS and IC

  4. The Best Theoretical Alternative for Real HCV • Similar virus to HCV • Easy to culture • Grows to high titers • Non infectious to humans • Detectable by HCV assays

  5. Viruses Related to HCV CONFIDENTIAL

  6. BVDV, HCV and MS2 CONFIDENTIAL

  7. HCV & BVDVGenome Comparison Target Region of NAT HCV assays BVDV genome is similar to HCV but can be easily cultured and manipulated

  8. Flaviviridae TranslationHCV & BVDV

  9. 5’UTR Secondary Structure • Critical 5’UTR secondary structure • Transcripts often do not have correct secondary structure • Might give misleading results for certain HCV genotypes • Is the Flaviviridae 5’-3’ Ring formation important?

  10. 5’ & 3’ NTR Strategies  SynTura

  11. Position of HCV 5’NTR in BVDV 5’HCV NTR NFAR

  12. Generation of a BVDV-Hybrid Make RNA Transcripts BVDV Plasmid (BVDV in blue) Insert desired Sequence (red) DNA RNA Viral Particles Transfect Cell Line Grow transfected Cell in culture Infected Cells produce Virus

  13. Development Timeline

  14. SynTura HCV Growth Curve

  15. Inactivation of BVDV • Inactivated with ß-Propiolactone • Protein-modifying agent • reacts with amides of Lys or Arg • Commonly used for BVDV vaccines • Alternative: Heat – 90% loss

  16. Initial Titer Determination

  17. Results with ProCleix Ultrio TMA Assay No impact of ß-PL inactivation on TMA Effect of Inactivation on TMA

  18. Sequence Stability • 3 passages 5 clones partially sequenced • No mutations found in 5 clones • 7 passages 5 clones partially sequenced • 1 clone no mutation • 2 clones had 1 point mutation in BVDV sequence • 2 clones had 1 point mutation in HCV insert • Rate of mutation in insert similar to virus

  19. SynTura Thermal Stability

  20. 37ºC Accelerated Stability

  21. SynTura HCV as Calibrator

  22. OptiQuant-S HCV RNA Quantification Panel • Based on SynTura Technology • 100, 500, 5e3, 5e4, 5e5, 5e6 & 2.5e7 IU/mL • Plasma Matrix

  23. Multicenter Study

  24. Roche CAP/CTM HCV IVD

  25. Comparison of two Abbott realTime HCV ASR Assays

  26. Key Results • SynTura HCV titer 2.0E+08 IU/mL • Concentrated to 1.5eE+10 IU/mL • Stable over 7 passages • Insert remains intact • No mutation after 3 passages • Only 1 point mutation after 7 passages

  27. Summary • SynTura has properties very similar to HCV and probably other enveloped mammalian viruses • SynTura technology allows integration of defined RNA sequences • SynTura technology can be used for RNA assays as: • QS and IC • Positive Control • Calibrator

  28. Martin Luther Universität Halle/Saale Sven Behrens Martina Behrens Acrometrix Mona Shahbazian Jerry Boonyaratanakornkit Reina Karunaratne Steve Young, Jessie Kilgore –Tricore Hanna Rennert, John Sipley, - NYPH, Cornell Medical Center Melody Hung-Fan, Kara Lee, -Contra Costa Public Health Lab Linda Sabatini, Lech Mazur, -ACL Central Lab Maura Pieretti, Carolyn Dowell, -BayCare Lab Ted Schutzbank- Covance Thank You

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