Precipitation steps
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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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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 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


Magnetic separation module i

Magnetic Separation Module I

Rod Head

Electromagnet

Tube track

Zentrallabor Hagen

SoGAT XVII, Paris, 27/05/04


Features

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


Materials

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


Workflow

Workflow

Zentrallabor Hagen

SoGAT XVII, Paris, 27/05/04


Sensitivity magnetic separation module i lightcycler ii abi sds 7700

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


Robustness

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


Outlook

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


Zentrallabor hagen

Thorsten Hageböck

Andrea Matulina

Yvonne Schmidt

Thanks to:

Zentrallabor Hagen

SoGAT XVII, Paris, 27/05/04


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