Post on 31-Jan-2016
description
DRK-Blutspendedienst West
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 .
Zentrallabor HagenZentrallabor Hagen
SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04
Magnetic bead technologyMagnetic bead technology
in viral RNA/DNA extraction from plasma minipoolsin viral RNA/DNA extraction from plasma minipoolsL. Pichl and V. SchottstedtL. Pichl and V. Schottstedt
German Red Cross Blood Transfusion Centre West, Central Laboratory, HagenGerman Red Cross Blood Transfusion Centre West, Central Laboratory, Hagen
DRK-Blutspendedienst West
Magnetic Separation Module I
SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04
Zentrallabor HagenZentrallabor Hagen
Rod HeadRod Head
Electromagnet
Tube track
DRK-Blutspendedienst West
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
SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04
Zentrallabor HagenZentrallabor Hagen
DRK-Blutspendedienst West
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 Kit
RealArt™ HAV LC RT PCR KitRealArt™ HBV TM PCR Kit
(www.artus-biotech.com)
Nucleic acid
Nucleic acid extraction
extraction
Amplification and
Amplification and
detection
detection
SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04
Zentrallabor HagenZentrallabor Hagen
DRK-Blutspendedienst West
Workflow
0 1h 2hrs 3hrs 4 hrs
Set up
Extraction
PAV B19
HAV
HBV
PCR
SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04
Zentrallabor HagenZentrallabor Hagen
DRK-Blutspendedienst West
SensitivityMagnetic Separation Module I + LightCycler II /ABI SDS 7700
Parameter PCR Cycler SpikeReferenceMaterial
95% detectionlimit*
[IU/ml single don.]
v.c. [%]
cp/threshold
PAV B19 LightCycler II WHO B19-DNA
NIBSC 99/800875 0.61 – 2.23
HAV LightCycler II WHO HAV-RNA
NIBSC 00/560260 0.45 – 2.76
HBV ABI SDS7700
WHO HBV-DNA
NIBSC 97/7461.274 1.02 – 2.71
* = calculated by probit analysis
Minipools of n = 93, EDTA-Plasma, triple-spike of diluted Ref. Mat., 100 µl each
SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04
Zentrallabor HagenZentrallabor Hagen
DRK-Blutspendedienst West
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.
SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04
Zentrallabor HagenZentrallabor Hagen
DRK-Blutspendedienst West
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.
SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04
Zentrallabor HagenZentrallabor Hagen
DRK-Blutspendedienst West
Thanks to:
Thorsten HageböckAndrea MatulinaYvonne Schmidt
Zentrallabor HagenZentrallabor Hagen
SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04