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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

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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

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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

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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

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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

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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

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1.66019 ~ 45.7 IU/ml (95%)

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1.15821 ~ 14.4 IU/ml (95%)

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1.12033 ~ 13.2 IU/ml (95%)

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New Automated Viral Sample Preparation

Link to Real-Time PCR Assays (1)

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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.

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Thank You

for Your Attention!