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Zentrallabor Hagen

Precipitation steps (PEG), centrifugation and careful resuspension of the invisible pelleted viral particles precede efficient extraction of viral RNA and DNA from minipools (n  96,  9.6 ml). Loss of nucleic acid during this time consuming steps is another critical point.

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Zentrallabor Hagen

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  1. Precipitation steps (PEG), centrifugation and careful resuspension of the invisible pelleted viral particles precede efficient extraction of viral RNA and DNA from minipools (n  96,  9.6 ml). Loss of nucleic acid during this time consuming stepsis another critical point. We looked for an automated RNA/DNA extraction method,that could start right from the minipool sample (n = 96, 9.6 ml). Release of labile blood components calls for short assay time . Magnetic bead technologyin viral RNA/DNA extraction from plasma minipoolsL. Pichl and V. SchottstedtGerman Red Cross Blood Transfusion Centre West, Central Laboratory, Hagen Zentrallabor Hagen SoGAT XVII, Paris, 27/05/04

  2. Magnetic Separation Module I Rod Head Electromagnet Tube track Zentrallabor Hagen SoGAT XVII, Paris, 27/05/04

  3. Features • Polyethanol coated magnetic beads (1 µm Ø, hydrophilic) • 12 Rod Head magnetizable • 12 samples, up to 10 ml vol.in 50 ml tubes • One specific disposable tip per sample per complete separation • Eluate volume: 100 µl • Processing time: 1h 8 min Zentrallabor Hagen SoGAT XVII, Paris, 27/05/04

  4. Chemagen Magnetic Separation Module I Chemagic Viral 10k Kit Special (www.chemagen.com) LightCycler II (Roche Diagnostics), ABI SDS 7700 RealArt™ ParvoB19 LC PCR KitRealArt™ HAV LC RT PCR KitRealArt™ HBV TM PCR Kit (www.artus-biotech.com) Materials Nucleic acid extraction Amplification and detection Zentrallabor Hagen SoGAT XVII, Paris, 27/05/04

  5. Workflow Zentrallabor Hagen SoGAT XVII, Paris, 27/05/04

  6. SensitivityMagnetic Separation Module I + LightCycler II /ABI SDS 7700 Minipools of n = 93, EDTA-Plasma, triple-spike of diluted Ref. Mat., 100 µl each Zentrallabor Hagen SoGAT XVII, Paris, 27/05/04

  7. 102 Pools have been analysed for B19, HBV and HAV so far. None of them tested positive. Cross contamination study was performed with two3 x 4 matrices of alternating negative and B19 spiked pools. Referring to a dose of 10EE 8 IU/ml per single donation all of the none spiked pool were negativein B19 LC-PCR. Tracing back reactive results from pools to single donation via ”chessboard“ testing was performedfor HAV, HBV and B19. Robustness Zentrallabor Hagen SoGAT XVII, Paris, 27/05/04

  8. Combination of Chemagen´s magnetic bead tech-nology with real time PCR without pre-extraction manipulation of the minipool (n = 96) reveals acceptable sensitivity and robustness for PAV B19, HAV and HBV; Implementation of a robotic sample processor with the magnetic separation module for sample identification, aliquoting reagents and sample lysis; Increasing throughput by optimizing time management. Outlook Zentrallabor Hagen SoGAT XVII, Paris, 27/05/04

  9. Thorsten Hageböck Andrea Matulina Yvonne Schmidt Thanks to: Zentrallabor Hagen SoGAT XVII, Paris, 27/05/04

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