ad_cm_ai_nrl-annual-meetings-13_pres-15.pdf

Biochemistry

1. 12.06.2007
1
Evaluation of automated RNA extraction
methods during an AI outbreak
Markus Durban
Friedrich Loeffler Institut

2. 12.06.2007
2
Outline
• Introduction
• Results of the evaluation of different extraction methods
and kits
• Summary

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3
Basics
• Real time PCR became more and more important in
diagnostics of viruses
• Ready-to-use kits for real time PCR are available
• Sensitivity of the assay is influenced by the yield and the
purity of the nucleic acid
• Nucleic acid extraction is now the most critical and labor-
intensive step

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More Basics
• Manual samplepreparationmethodsare
– labor intensive
– susceptibleto contaminations
– susceptibleto handlingvariations
– susceptibleto errors
• Automatedextractionmethodsareneededforhigher
amountsof samples

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Closed systems
•Positive
– Validated methods for use in clinical diagnostics
– Easy to handle
– Short training period
• Negative
– Kits and plastics mostly offered by one supplier
– Mostly long durations for extractions
– Only one extraction method can be used

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Open platforms
•Positive
– Free in choosingthe extraction method and the kit
– Optimization of the extractionprotocols possible
– The use of different methods and kits for different
problems is possible
• Negativ
– Mostly no validated methods
– Longer training period
– Programming is necessary

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7
Configuration of our extraction robot
Pipetting arm
Barcode-Reader
Gripper
Troughs for buffers
Cooled rack
Vacuum station
Waste and washing station
Lysed samples
Magnetic separator
Heater

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Silica membranes
• Nucleic acids are bound at silica membranes
• Removal of liquids by vacuum or centrifugation

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• AIV H12N5-dilution curve 10
-1
to 10
-6
Macherey& Nagel NucleospinMulti 96 Virus
38,1/no Ct no Ct 10
-7
36,1 36,8 10
-6
34 33,9 10
-5
30,6 29,4 10
-4
27,1 26,5 10
-3
20,3
23,5
-
22,7
10
-1
10
-2
NucleoSpin
Multi 96 Virus [Ct]
QIAamp Viral
Mini Kit [Ct] Dilution step

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• AIV H12N5-dilution curve 10
-1
to 10
-6
Macherey& Nagel NucleospinMulti 96 Virus
FAM HEX

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11
• Validated for RNA-extraction from liquids for:
- Avian influenza (AIV)
- Newcastle disease (NDV)
- Foot and mouth disease (FMDV)
- Swine vesicular disease (SVD)
- Classical swine fever (CSF)
- Bluetongue (BTV)
Macherey& Nagel NucleospinMulti 96 Virus

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Macherey & Nagel Nucleospin Multi 96 Virus
• RNA extraction using Trizol as lysis reagent
- Samples are mixed with Trizol
- Chloroform is added and the samples are centrifuged
- The upper phase is used for further extractions

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• Problems with:
- Swabs→none
- Blood serum →sporadic clots
(partial with Trizol)
- EDTA-Blood →sporadic clots
(1:1 diluted with PBS)
- Tissue →sporadic clots
(with Trizol)
Macherey& Nagel NucleospinMulti 96 Virus

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QIAamp Virus BioRobot MDx Kit
• AIV H12N5-dilution curve 10
-1
to 10
-6
FAM HEX

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• AIV H12N5-dilution curve 10
-1
to 10
-6
Zymo ZR-96 Viral RNA Kit

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• AIV H12N5-dilution curve 10
-1
to 10
-6
Invisorb Virus RNA HTS 96 Kit/ep
FAM HEX

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17
Summary for Kits based on silica membranes
37.91 35.50 35.78 36.74 10
-6
34.91 34.31 33.04 34.01 10
-5
31.86 34.02 29.89 30.06 10
-4
28.40 31.36 26.59 27.40 10
-3
25.02 25.90 23.33 23.46 10
-2
21.61 22.30 20.09 19.54 10
-1
Invisorb Virus
RNA HTS 96
Kit/ep
Zymo ZR-96 Viral
RNA Kit
QIAamp Virus
BioRobot MDx Kit
Macherey&Nagel
Nucleospin Multi
96 Virus

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Magnetic beads
• Nucleic acidis bound on coated magnetic particles
• Particles are washed in tips or cavities
Separation in
the tip
Separation on a
magnet
Separation in
the cavity

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19
• Using the BioRobot M48
• AIV H12N5-dilution curve 10
-2
to 10
-6
QIAGEN MagAttractVirus Mini M48 Kit
+ Good results already in the
first run
+ Validated for clinical
diagnostics
- Almost 5 h for 48 Samples

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20
• Using the Tecan freedom evo
• AIV H12N5-dilution curve 10
-1
to 10
-6
QIAGEN MagAttractVirus Mini M48 Kit
Eluate incubated for 10 min at 70°C

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21
• Using the Chemagen MSM 1
• AIV H12N5-dilution curve 10
-1
to 10
-6
Chemagic Viral DNA/RNA Kit
FAM HEX

22. 12.06.2007
22
• Using the Tecan freedom evo
• AIV H12N5-dilution curve 10
-1
to 10
-6
Chemagic Viral DNA/RNA Kit

23. 12.06.2007
23
• Using the Tecan freedom evo
• AIV H12N5-dilution curve 10
-1
to 10
-6
Macherey & Nagel NucleoMag 96 Virus Kit

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• Using the Tecan freedom evo
• AIV H12N5-dilution curve 10
-1
to 10
-6
AGOWA mag Maxi DNA Isolation Kit

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Summary for kits based on magnetic beads
38.19 38.39 37.81 38.47 37.08 37.65 10
-6
37.36 36.05 33.86 37.18 35.93 37.56 10
-5
34.55 33.54 30.56 33.38 33.56 33.27 10
-4
31.73 29.72 27.18 30.05 30.56 29.39 10
-3
28.38 26.67 23.74 26.81 26.97 25.55 10
-2
24.83 23.07 19.71 23.08 - 22.07 10
-1
AGOWA mag
Maxi DNA
Isolation Kit
NucleoMag 96
Virus Kit
Chemagic
Viral
DNA/RNA Kit
(on MSM 1)
Chemagic
Viral
DNA/RNA Kit
MagAttract
Virus Mini
M48 Kit
(on M 48)
MagAttract
Virus Mini
M48 Kit

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26
• Extracted until now:
- around 30.500 samples of dilution curves, animal
experiments and diagnostic samples
~14.300 AIV/NDV samples
~9.300 BTV-EDTA-blood samples
~1.300 serum samples
~2.000 samples with trizol as lysis buffer
~2.100 tissue samples
~2.000 different samples for kit evaluation
SummaryI

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• The automated RNA extractions from liquid samplesis
actually done with NucleoSpin Multi 96 Virus Kit
• QIAamp Virus BioRobot MDx Kit, MagAttract Virus Mini M48
Kit and Chemagic Viral DNA/RNA Kit can be used as
alternative
• The extraction from liquids is validated for AIV, NDV,FMD,
SVD, CSF, and BTV
• A combination of the used kit with trizol als lysis buffer is
possible and established for serum, blood and tissue
samples
• The extraction of virus RNA in tissue is almost validated for
CSF, AIV and NDV using the NucleoSpin 96 RNA Tissue Kit
• In case of an outbreak more than 1000 samples can be
extracted and analyzed per day
SummaryII

28. 12.06.2007
28
Thank You
Martin Beer
Bernd Haas
Bernd Hoffmann
Ulrike Polenz
Christian Korthase
Anja Schulz
Federal Ministry of Food, Agriculture and Consumer Protection
…and all others for bringing me samples