1. Tom Øystein Jonassen, Mona Holberg-Petersen, Einar S. Berg
Assessment of the reliability of nucleic acid extraction systems commonly used to get valid qPCR results
Tom Øystein Jonassen, Mona Holberg-Petersen, Einar S. Berg

2. Problem:
Which extraction platform to use by the detection of a certain target organism?
Sensitivity – one NA molecule alone
IVD/CE-labeling
Sensitivity – one NA molecule among 1012 molecules
Suited for WGS
Flexibility 8 – 96 samples
Cost instrument
Flexibility - various input materials
Cost reagents/kits
Easy operation
Flexibility - various input volumes
Cost consumables
Automatic operation
Flexibility - various output volumes
Rapid processing
Cost service/maintenance
External lysis

3. Typical logistics by NA diagnostic testing
Contained
internal control
nucleic acid
Specimens
collection
Transport
Sample-
preparation
”Naked”
internal control
nucleic acid
Signal
generation
Real-time
PCR
Detection

4. Experiment design – the DNA/RNA extraction in focus
WHO
International
Standards
Specimens
collection
Transport
Sample-
preparation
Real-time
PCR
Signal
generation
Detection

5. GenoSen’s real-time PCR kits

6. QS’s of different run of HBV assay QS’s of different run of HCV assay
Test reliability
QS1: 1x105 IU/µl
QS5: 1x101 IU/µl
GenoSen’s provided QS’s:
QS’s of HBV assay
QS’s of HCV assay
QS’s of different run of HBV assay
QS’s of different run of HCV assay Ct

7. Aliquots of DNA virus material Aliquots of RNA virus material
Standardized samples
WHO International
Standards
Diluent: EDTA plasma from
registered blood donors S1 1x S2
0,5x S3
0,1x
50x:
HBV; 1,0 x 106 IU/ml
HCV; 1,6 x 105 IU/ml
HIV-1; 7,3 x 105 IU/ml S4
0,05x S5
0,01x S6
NEG
Aliquots of DNA virus material S1 S2 S3 S4 S5 S6
Aliquots of RNA virus material

8. The IC particles invention
Internal QC, IC initially spiked in EDTA-plasma
The IC particles invention
Liposome-based cell/virus mimicking vehicle for IC’s of NATs, tailored for whole process Quality Control

9. Freeze/thawing procedure
Preparation of IC particles
Freeze/thawing procedure
Small unilamellar vesicles (SUV)
Extrusion
Freeze/
thawing
Add
nucleic acid
Large unilamellar vesicles (LUV)
Multilamellar vesicles (MLV)
Sonication
Vortex
Add buffer
Dried lipid film
Swelling

10. In the spotlight; the extraction platforms
BioRobot EZ1
QIAcube
QIAamp Viral RNA
QIAamp DSP
NucliSENS easyMAG
QIAamp DNA mini
COBAS AmpliPrep
Roche HighPure NA
bioMerieux NucliSens
Affigene
MagNA Pure Compact
MagNA Pure LC

11. Geno-Sen’s HBV, HCV, HIV-1
Experiment logistics
IC particles (ICDNA)
HBV – DNA virus 1x
0,5x
0,1x
0,05x
0,01x
NEG
Aliquots of diluted
WHO standards
HCV – RNA virus
HIV – RNA virus
IC particles (ICRNA)
3x200µl plasma input
QIAamp DNA mini
(manuall)
QIAamp Viral RNA
(manual)
EZ1 Virus Kit 2.0
(robot)
Magnapure LC
(robot)
Ampliprep
(robot)
Easymag
(robot)
Affigene DNA
extraction
(manual)
QIAamp Viral RNA
(QIAcube-robot)
QIAamp DNA mini
(QIAcube-robot)
QIAamp DSP
(manual)
Roche High pure NA
(manual)
NucliSens
(manual)
Magnapure Comp
(robot)
3x100µl elution output, 2x5µl PCR input
CAS1200 robot pipetting
Geno-Sen’s HBV, HCV, HIV-1
realtime PCR assays

12. RESULTS: Which extraction platform to use by the detection of
i) HBV - a DNA virus?
ii) HCV or HIV-1 - RNA viruses?

13. HBV HBV signal IC signal QIAamp DNA Mini, manual QIAamp DNA Mini, Cube
QIAamp DSP
BioRobot EZ1, Virus
Affigene
Roche
HighPure
AmpliPrep
MagNa Pure Compact
MagNa Pure LC
tot NA
Nucli Sens, manual
Nucli Sens, EasyMag
Roche
HighPure
HBV signal
IC signal
QIAamp DNA Mini, Cube
Roche
AmpliPrep
QIAamp DNA Mini, manual
MagNa Pure Compact
QIAamp DSP
MagNa Pure LC
tot NA
BioRobot EZ1, Virus
Nucli Sens, manual
Affigene
Nucli Sens, EasyMag

14. qPCR data of hepatitis B virus DNA extracted by various platforms
HBV
qPCR data of hepatitis B virus DNA extracted by various platforms
Average
ICDNA signal

15. HCV HCV signal IC signal HCV signal IC signal Roche AmpliPrep crash
extractor
QIAamp Viral RNA, manual
QIAamp Viral RNA, Cube
QIAamp DSP
QIAamp DNA EZ1
HighPure
MagNa Pure Compact
MagNa Pure LC,
tot NA
Nucli Sens, manual
Nucli Sens, EasyMag
QIAamp Viral RNA, Cube
Roche
AmpliPrep
crash
extractor
QIAamp Viral RNA, manual
MagNa Pure Compact
QIAamp DSP
MagNa Pure LC,
tot NA
QIAamp DNA EZ1
Nucli Sens, manual
Roche
HighPure
Nucli Sens, EasyMag

16. qPCR data of hepatitis C virus RNA extracted by various platforms
HCV
qPCR data of hepatitis C virus RNA extracted by various platforms
Average
ICRNA signal

17. HIV-1 HIV-1 signal HIV-1 signal No IC signal; liposomal IC target
not compatible with
HIV-1 RT-PCR
HIV-1 signal
Roche
AmpliPrep
extractor crash
QIAamp DNA Mini, Cube
QIAamp DNA Mini, manual
QIAamp DSP
QIAamp DNA EZ1
HighPure
MagNa Pure Compact
MagNa Pure LC
tot NA
Nucli Sens, manual
Nucli Sens, EasyMag
QIAamp DNA Mini, Cube
Roche
AmpliPrep
extractor crash
MagNa Pure Compact
QIAamp DNA Mini, manual
MagNa Pure LC
tot NA
QIAamp DSP
Nucli Sens, manual
QIAamp DNA EZ1
Nucli Sens, EasyMag
Roche
HighPure

18. qPCR data of HIV-1 RNA extracted by various platforms

19. Affigene DNA extraction
HBVDNA
Affigene DNA extraction
Qia DNA mini, man
COBAS Ampliprep
Qia DNA mini, cube
BioRobot EZ1
HIV-1RNA
MagNA pure, LC
Roche HighPure
And the winner is?
Qia DSP
MagNA pure, Comp
HCVRNA
Qia V-RNA, cube
Qia V-RNA, man
NucliSens
Easymag

20. Contributors IC Particles AS Acknowledgements: Synnøve Hestad Myhre
Hege Fremstad
Bjørg Guri Gutigard
Norwegian Institute of Public Health
ULLEVAAL
University Hospital
Gunilla Lövgården
Marie Elisabeth Vad
IC Particles AS
The Research Council
of Norway