nucleic acid isolation for diagnostic testing using bayer´s magnetic · pdf...
TRANSCRIPT
Dr. Guido HennigBayer HealthCare AGDiagnostics Research Germany
2nd qPCR Symposium, Freising, [email protected]
Nucleic Acid Isolation for
Diagnostic Testing using
Bayer´s Magnetic Particles
Bayer HealthCare Divisions
AnimalHealth
BiologicalProducts
ConsumerCare
Diagnostics
Pharma
Diabetes Care
Diagnostics
Laboratory Testing
Near Patient Testing
Molecular Testing
Diagnostics Research,Leverkusen
Slide 3
Pre-Analytical Steps: Sample Preparation
� Efficient isolation of nucleic acids is a key prerequisite
for their qualitative and quantitative detection
in diagnostic applications
� Pure nucleic acids / Interference free
� Process automation
� Sensitive with high recovery/purification efficiency
Clinical Need for Technical
Performance
Slide 4
History of Nucleic Acid IsolationIn
no
vatio
n
� Column Technology (Silica particles)– Research (automation difficult)
� Phenol / Chloroform– Special case
� Silica-coated magnetic particles - In Vitro Diagnostic
Slide 5
Advantages of Magnetic Particle Technology
� Simple and low-cost automation
� High reproducibility
� High sample throughput
� Flexible in its applications
� Scalability of sample volume
Slide 6
Bayers „new“ Magnetic Particles
� „One particle chemistry“ for all applications in the field of nucleic acid
isolation
� Coating of iron oxides with nanolayer of silica
� Simple, robust and controlled manufacturing process
Others
� No change in morphology
Slide 7
� „homogenous“ particle size distribution Reproducibility
PropertiesProperties Automation AdvantageAutomation Advantage
� small particles (< 1 µm) optimal suspension behaviour
� very high iron content efficient magnetization
� very good paramagnetism efficient isolation
� high storage stability Reliability
Properties Determine Performance
Slide 8
Nexsys Assay Development
Kinetic Amplification Assays in Development
• HCV (RNA) currently the assay has a sensitivity of 10 copies/reaction(genotypes 1 through 6)
• HIV (RNA) current level of sensitivity is 10 copies/reaction and(subtypes of group M and group O)
• HBV (DNA) current level of sensitivity is 10 copies/reaction
• CT/GC (DNA) current level of sensitivity is 10 copies/reaction
Infectious Disease Testing Assay Portfolio
Schematic Nucleic Acid Isolation Protocol
Mag
net
Elution
Release
Detection and Quantification with kinetic Amplification
Chaotropic binding buffer
Proteinase K
Magnetic particle
PlasmaM
ag
net
WashM
ag
net
Supernatant
Mix
Incubation
Binding
Eluate + MasterMix
Automation of HCV Isolation (Model System)
� Commercial Liquid Handling Robot
� Module
� Samples
� Reagents
� Tips
� Waste
� Microtiter plate
� Shaker
� Heating block
� Magnet
� Complete automation
Slide 11
Set-Up Flexibility
� Automated protocol allows flexible purification
of different sample batches
8, 16, 24 ... - 96 samples (< 2,5 hours for 96)
� Automated protocol allows flexible purification
of different sample volumes
500 µl or 1 ml
Slide 12
Panel Number
Concentration
(copies/mL)
LP1 10,000,000
LP2 1,000,000
LP3 100,000
LP4 10,000
LP5 1,000
LP6 100
LP7 50
LP8 25
Negative Serum 0
Negative Plasma 0
Panel StudyHCV Samples for Performance Study
� HCV plasma samples
were value-assigned
with quantitative
VERSANT ® HCV
RNA assay
(bDNA)
Slide 13
Specificity and Imprecision
� 100% Specificity
No false positives could be detected
when copurified with neighbouring
high positive samples
� Good Reproducibility
� within run
� between run
CV´s < 25% over most of the dynamic range
Slide 14
Linearity and Sensitivity
� Linearity
at least 5 magnitudes
from 107 down to 102 HCV copies/ml
y = 0.989x - 0.15
R2 = 0.99
2
3
4
5
6
7
2 3 4 5 6 7
Log concentration detected
log
in
pu
t c
on
ce
ntr
ati
on
� Sensitivity / LOD (95% hit rate)
with 500 µl Plasma
55 HCV copies / ml (11 IU / ml)
� Recovery / Purification Efficiency 80 - 100 %
Slide 15
Universal Applicability
Nucleic Acid Purification protocols designed for
� Pharmacogenomic DNA Testing
� Cystic Fibrosis Testing
� Predictive SNP´s for Cardiovascular Risk Assessment
Sample: EDTA Blood
� Oncology Testing
� Predictive RNA Marker Panels
for Individualized Therapy
Sample: Fixed Paraffin-Embedded (FPE) Tissue
Slide 16
Benchmark DNA Isolation from Blood
� Manual Isolation from
100 µl EDTA blood
showed comparable
yields for Bayer and
two commercial kits
Benchmark 100 µl Blood
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Bayer Method Kit A (bead) Kit B (column)
DN
A y
ield
in
ng
Pico
PCR
Comparison Bayer vs Kit (automated)
0
1000
2000
3000
4000
5000
6000
#1 #2 #3 #4 #5 #6Frozen Sample No.
Yie
ld D
NA
in
ng
Bayer System
Bead Based Kit
� Automated Side to Side
Benchmark on standard liquid
handling platform showed much
better yields with Bayer method
compared to commercial bead kit
Slide 17Archived FPE tissue RNA is fragmented (100-500 bp)
RNA Fragment Size Bayer vs. Column Kit
M Control:
MCF-7 fresh frozen RNA
Kit ABayer Bead
0.2 kb
0.5 kb
1.0 kb
2.0 kb
4.0 kb
6.0 kb
28S
18S
Mean size Kit A
Mean size Bayer
8 consecutive slides
from 1tumour block
Slide 18
Yield of Total RNA from Archived FPE Slides
Expression profiling of predictive/prognostic marker panels
with qRT-PCR on archived FPE tissue RNA feasible
RNA from about 271 clinical breast cancer samples (10 µm slides
up to 8 years old) was isolated with manual Bayer protocol
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
0
10
20
30
40
50
total RNA yield in µg (RiboGreen)
Fre
qu
en
cy
• Failure Rate: < 1 %
• Low yield ( < 500 ng): < 10% (22 of 271)
Slide 19
Summary
� Robust and new Bayer magnetic particles
� Essential part of Bayer´s detection platforms
� Sensitive detection assays for HCV, HIV, HBV and CT/GC
� Efficient and quantitative procedure
for automated isolation of HCV + HIV
� Flexible in its applications for
� Infectious disease testing
� Pharmacogenomics
� Tumour diagnostics
Guido Hennig Karlheinz Hildenbrand
Heike Paus Helmut Krülls
Torsten Acht Dirk Mangold
David Sherman
Christoph Petry
Ralph Wirtz
Udo Stropp
Acknowledgements