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Molecular Automation
450 444 Autoanalysers in Clinical Laboratory Viraphong Lulitanond, Ph.D. g@ viraphng@kku.ac.th
Why Wh
Molecular techniques ?
Molecular Automation
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Molecular Automation
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Antibodies are detectable approximately 80 days after infection. Viral RNA is detectable approximately 23 days after infection.
HCV RNA
Ab to HCV 0 10 20 30 40 50 60 70 80 90
Molecular Automation
1998
Molecular Automation
PCR Process
1. Sample preparation (Pre-PCR) 2. Amplification (PCR) 3. PCR products detection (Post-PCR)
Gel electrophoresis Hybridize with specific probe Plate-based hybridization assay y y Sequencing
Molecular Automation
Taq Thermus aquaticus Vent, Deep Vent Pfu Pyrococcus furiosus Tth Thermus thermophilus
Conventional PCR
Separate step of amplification and detection End point detection in the plateau phase of the reaction Take 2-3 hours for amplification Low throughput Detection by : hybridization
blotting gel electrophoresis
Narrow dynamic range Open tube format (in-house) Easy false contamination (in-house)
Molecular Automation
Molecular Automation
1998
B Blood Screening
Microbiology
Genomics
Oncology
Virology
Molecular Automation
Degree of Automation
Molecular Automation
Semi-automation system
AMPLICOR assay Launch in 1992 First ready to use Standradized kit
TC 9600
COBAS AMPLICOR
Launch in 1995 First fully automated amplification and detection Second generation assays, both qualitative and quantitative assays
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Molecular Automation
COBAS AMPLICOR
Photometer
Computer
Printer
Incubator
Wash station
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Molecular Automation
COBAS AmpliPrep
COBAS AmpliPrep system processes clinical specimens Generate about 24 processed samples per hour or up to 144 samples per day Process serum or plasma RNA specimens Random Access analyzer
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Molecular Automation
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Molecular Automation
SYBR Green I
Elongation Phase
Annealing Phase
Elongation Phase
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Molecular Automation
Cycle Number
Template: Plasmid; Target: TNF; Detection Format: SYBR Green I
Fluorescence -d (F1) / dT
1E+1
Fluorescence (F1)
Temperature C
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Molecular Automation
Denaturation: Double strand DNA to Single strand DNA by heat. No FRET Probe hybridization : two oligo probe hybridize to DNA strand. Fluorescent signal can be monitored Elongation : Strand displacement by primer elongation
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Molecular Automation
COBAS TaqMan 48
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Molecular Automation
COBAS TaqMan 48
Size Weight Batch Size Channels Disposable (45 x 75 x 50) cm 53kg 2 x 24 (50 100 l) 4 (520 nm, 575 nm, 645 nm, 725 nm) Plastic tube (k-tube) modified k-tray Automation Manual transfer required
Benefit
Broad dynamic range No separate detection Assay time reduction Increases Throughput Provides internal control Eliminates contamination risk Allows multiplexing Will allow additional formats (Hybridization Probes) Easy handling Automation through AmpliPrep
Integrated Quantification Standard requires no external standard curves Closed Tube Format Four color channels
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Molecular Automation
TC lid
TC cover
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Molecular Automation
Melting curve analysis Potential to run Hydrolysis Probe, Hybridization probe, Beacon probe, Scorpian probe and others. Customer can use system for own applications
COBAS TaqMan 48
COBAS TaqMan 96
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Molecular Automation
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Molecular Automation
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Molecular Automation
LightCycler SystemFeatures
100l / 20l Capillaries Multicolor Detection / Multiplex-PCR
Mutation & SNP Detection Elimination of Contamination Risk Research & Food Safety Rapid Real-Time PCR
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Molecular Automation
LightCycle Accessories
LightCycler Capillaries: 100l and 20l LightCycler Carousels for 100l and 20l Capillaries
Disposable :
Reaction volume : 20, 100 uL Software : S ft Light C l Li ht Cycler software f analysis and i t ft for l i d interpretation t ti of realtime quantification and melting curve data IVD kit and research application
Utility :
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Molecular Automation
Speed
Flexibility
Safety
Reliability
Success
Components
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Molecular Automation
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