1 / 9

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.

ziazan
Download Presentation

Zentrallabor Hagen

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related