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

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

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

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

GenoSen’s real-time PCR kits

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

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

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

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

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

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

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

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

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

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

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

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

qPCR data of HIV-1 RNA extracted by various platforms

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

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