nucleic acid isolation for diagnostic testing using bayer´s magnetic · pdf...

Dr. Guido HennigBayer HealthCare AGDiagnostics Research Germany

2nd qPCR Symposium, Freising, 2005-09-05guido.hennig@bayerhealthcare.com

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