Upload
clementine-ball
View
213
Download
0
Tags:
Embed Size (px)
Citation preview
W W W . Q I A G E N . C O M
Generic Automated Sample Preparationfor Commercial NAT Assays
Markus Sprenger-Haussels, PhD
Associate Director R&D
QIAGEN GmbH, Hilden, Germany
14.06.2006 SoGAT XIX, Bern, Switzerland
W W W . Q I A G E N . C O M
Automation of Human Sample Preparation
1 6 48 96 x 96 # samples
BioRobot EZ1
Magnetic Beads
BioRobot M48
Magnetic Beads
BioRobot MDx
Silica Membranes
Protedyne BioCube
Membranes / Beads
Chemistry + Application Expertise + Instrumentation
W W W . Q I A G E N . C O M
Viruses are different
single stranded double stranded RNA Viruses DNA Viruses linear genome circular genome degree of secondary structure enveloped or not lysis behavior low to high copy no. NA heat labile or not
Different assays
artus® Assays Roche Amplicor®
Roche Cobas®TaqMan Abbott LCx®
Abbott Taqman Bayer bDNA Bayer TMA Innogentics InnoLipa Homebrew many other
Starting material is different
liquid samples (plasma, serum, CSF, urine) solid samples (tissue, swabs, blood cards, stool) mechanical forces needed for disruption enzymatic forces required thermal lysis required chemical lysis sufficient rich in protein rich in polysaccharides rich in salts rich in everything rich in inhibitory metabolites interfering medical treatment
Parameters Influencing NAT Assays
Assay/Detection Preanalytical
Different controls
“naked” RNA amored RNA plasmids
linear DNA armored DNA virus like particles different length
W W W . Q I A G E N . C O M
Example: Magnetic Bead Technology
Technology used for automation
High number of optional modifications Size Weight Core material Surface material Surface structure Movement in liquids Physical change by protocol steps
Differential binding of different nucleic acid types
W W W . Q I A G E N . C O M
Various Assays used during Development:
artus Assays
HBV*, EBV*, HSV*, HAV*, CMV*
other artus Assays
QuantiTect PCR Kits
Homebrew Virus Assays
Other Commerical Assays
Roche Assays
Abbott Assays
Bayer Assays
Others
BioRobot EZ1
BioRobot M48
New Automated Viral Sample PreparationLarge Number of Assays covered
* as CE marked IVD only available in Europe
W W W . Q I A G E N . C O M
Rotor-GeneTM
14.4 IU/ml
LightCycler®
45.7 IU/ml
TaqMan®
13.2 IU/ml
EZ1 DSP Virus Kit with artus® HBV PCR Kits (CE)*
95 % probit values on different detection platforms
13 different dilution levels; 18 replicates per level
* artus HBV PCR Kits only available in Europe as CE-IVDs; EZ1 DSP Virus Kit under CE-IVD development
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
-2 -1 0 1 2 3 4log (dose)
prop
ortio
ns
1.66019 ~ 45.7 IU/ml (95%)
95%
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
-3 -2 -1 0 1 2 3 4log (dose)
prop
ortio
ns
1.15821 ~ 14.4 IU/ml (95%)
95%
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
-3 -2 -1 0 1 2 3 4log (dose)
prop
ortio
ns
1.12033 ~ 13.2 IU/ml (95%)
95%
New Automated Viral Sample Preparation
Link to Real-Time PCR Assays (1)
W W W . Q I A G E N . C O M
New Automated Viral Sample Preparation
Link to Real-Time PCR Assays (2)
EZ1 DSP Virus Kit* with other NAT Assays:
first verification LOD data with limited replicates
*Under development for in-vitro-diagnostic use in Europe, not available in the U.S.A.
W W W . Q I A G E N . C O M
Thank You
for Your Attention!