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Molecular Automation

Molecular Automation
450 444 Autoanalysers in Clinical Laboratory Viraphong Lulitanond, Ph.D. g@ viraphng@kku.ac.th

Research and Diagnostic Center for Emerging Infectious Diseases

Why Wh
Molecular techniques ?

Viraphong Lulitanond, Ph.D.

Molecular Automation

The Better Choices


Increase diagnosis accuracy Diagnose earlier Provide prognosis Predict risk of disease Reduce costs per diagnosis Reduce length of hospital stay

Nucleic acid testing utility in laboratory practice


opportunity t it
genetics and cancer

blood bank screening g infectious diseases

time

Viraphong Lulitanond, Ph.D.

Molecular Automation

Narrowing The Window On Transfusion-Associated HIV-1 Infection


Antibodies are detectable approximately 22 days after infection. p24 Ag is detectable approximately 17 days after infection. Viral RNA is detectable approximately 11 days after infection.
Viral RNA, Ag or Ab Level HIV-1 RNA p24 Ag Ab to HIV-1

10

20

30

40

50

60

70

80

90

Days post infection

Narrowing The Window On Transfusion-Associated HCV Infection

Antibodies are detectable approximately 80 days after infection. Viral RNA is detectable approximately 23 days after infection.

Viral RNA or Ab Level

HCV RNA

Ab to HCV 0 10 20 30 40 50 60 70 80 90

Days post infection

Viraphong Lulitanond, Ph.D.

Molecular Automation

Advantage of Nucleic Acid Amplification Techniques


Less amounts of samples Sensitivity Specificity Rapid Simple Offer - Diagnosis (Qualitative) - Prognosis (Quantitative) - Therapeutic Monitoring (Quantitative)

PCRIn the past

1998

Viraphong Lulitanond, Ph.D.

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

THERMUS AQUATICUS FROM YELLOWSTONE NATIONAL PARK

Source of Taq DNA polymerase

Viraphong Lulitanond, Ph.D.

Molecular Automation

Thermostable DNA Polymerase

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)

Viraphong Lulitanond, Ph.D.

Molecular Automation

ETHIDIUM BROMIDE STAINING UNDER UV ILLUMINATION

Molecular i h M l l weight marker k

Programmable Temperature Cycler (PCR machine)

Viraphong Lulitanond, Ph.D.

Molecular Automation

Roche Molecular Diagnostics

1998

Roche Molecular Diagnostics

B Blood Screening

STD & Viral O Oncology (HPV)

Microbiology

Viraphong Lulitanond, Ph.D.

Genomics

Oncology

Virology

Molecular Automation

COBAS AMPLICOR Test Profile


HIV-1 RNA (viral load) HIV HCV RNA( i l l d) RNA(viral load) HBV DNA(viral load) CMV DNA(viral load) AmpliScreen HIV AmpliScreen HCV AmpliScreen HBV CYP 450 HIV-1 proviral DNA HIV HPV HCV qualitative HCV genotyping C. trachomatis N. gonorrhoeae M. tuberculosis Beta thalassemia

Evolution of Roche Diagnostic Platforms


COBAS TAQMAN Integrated Walk-away System COBAS AMPLIPREP Automated Sample Preparation

Degree of Automation

AMPLILINK COBAS AMPLICOR MONITOR

COBAS AMPLICOR MWP


1992 1995 1996 2001 2003

Viraphong Lulitanond, Ph.D.

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

Viraphong Lulitanond, Ph.D.

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Molecular Automation

COBAS AMPLICOR
Photometer

Computer

Printer

COBAS AMPLICOR System


Amplification and Detection
Load samples, run information and reagents, start COBAS

AMPLICOR and walkaway


Parallel mode: load 2nd set of specimens after amplification of 1st Pipettor Photometer

Thermal cycler B Detection positions Thermal cycler A

Incubator

Reagent racks D-cups

Wash station

Viraphong Lulitanond, Ph.D.

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Molecular Automation

COBAS AmpliPrep Automation Sample Preparation


Minimum instrument set up Mi i i Full automation Flexible Continuous operation: Additional samples Onboard storage of prep. samples Overnight operation

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

Viraphong Lulitanond, Ph.D.

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Molecular Automation

Real Time PCR


hSimultaneous amplification and detection ( no separate detection ) hKinetic quantification in the log phase of the reaction hData is collected at each cycle and are displayed immediately y p y y

Common Features and Benefits


hQualitative and / or Quantitative analysis hMutation Analysis / SNP hReduce assay time hIncrease throughput I th h t hBroad dynamic range hClose tube format hEliminate contamination risk

Monitoring of PCR Reactions


Y = X(1+E)n

Viraphong Lulitanond, Ph.D.

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Molecular Automation

PCR and the Problem of Quantification


loglog-phase analysis
end-p end-point analysis y high concentration / high efficiency high concentration / low efficiency low concentration / high efficiency N : number of amplified molecules n : number of amplification cycles

Main problems of PCR: 1. Plateau effect 2. Efficiency of amplification

Real-time PCR Detection Formats

SYBR Green I

Hybridization Probe Format

Hydrolysis Probe Format

Elongation Phase

Annealing Phase

Elongation Phase

Viraphong Lulitanond, Ph.D.

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Molecular Automation

SYBR Green l Format Overview


D Denaturation: Double strand t ti D bl t d DNA to Single strand DNA by heat Primer Annealing: SYBR Green I start binding to minor grove of DNA strand Elongation : SYBR Green I has maximum Fluorescent signal can be monitored

Monitoring of PCR with the LightCycler using SYBR Green I


H2O 1E+2 1E+3 1E+4 1E+5 1E+6 1E+7 1E+8 1E+9 1E 9

Cycle Number
Template: Plasmid; Target: TNF; Detection Format: SYBR Green I

Fluorescence -d (F1) / dT

1E+1

Fluorescence (F1)

Temperature C

Viraphong Lulitanond, Ph.D.

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Molecular Automation

Hybridization Probes Format Overview


Fluorescein LC Red

transfer excite emission

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

LightCycler Fluorescence Excitation and Emission Spectra


Excitation Fluorescein Emission Fluorescein Excitation Red 640 Emission Red 640 Excitation Red 705 Emission Red 705

Viraphong Lulitanond, Ph.D.

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Molecular Automation

Hydrolysis Probe Format Overview


reporter
quencher

COBAS TaqMan 48

Viraphong Lulitanond, Ph.D.

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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

COBAS TaqMan Analyzers Common Features and Benefits


Feature
Kinetic PCR

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

Sample vessel: k-tube or k-plate

Viraphong Lulitanond, Ph.D.

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Molecular Automation

COBAS TaqMan Analyzer Thermal cyclers

TC lid

TC cover

Two independent thermal cyclers for maximum flexibility

COBAS TaqMan 48 2 Applications


IVD (HIV, HCV, HBV, HPV, HSV, CT) Utility Channel (in house)

Viraphong Lulitanond, Ph.D.

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Molecular Automation

Cobas TaqMan 48 - Utility Channel


Two independent Thermal Cyclers
two different protocols simultaneously two independent runs possible

Melting curve analysis Potential to run Hydrolysis Probe, Hybridization probe, Beacon probe, Scorpian probe and others. Customer can use system for own applications

Total PCR Automation


COBAS AMPLICOR

COBAS TaqMan 48

COBAS TaqMan 96

Viraphong Lulitanond, Ph.D.

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Molecular Automation

Automated Sample Preparation


MagNA Pure LC MagNA Pure Compact Benefit ..
Increase precision of every pipetting step High reproducibility quality and quantity of DNA, RNA, mRNA Reduce risk from manual sample handling

MagNA Pure LC System


True walk - away automation for nucleic acid purification l i id ifi i 1- 32 samples per run (within 1 hr.) Automated PCR set-up in different formats ( eg. LC capillaries , COBAS A-ring or 96-well plates). Broad variety of sample materials (blood, plasma, serum, sputum, tissue, feace, etc.)

Viraphong Lulitanond, Ph.D.

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Molecular Automation

MagNA Pure Compact System


True walk - away automation for nucleic acid purification 1-8 samples per run (within 30 min) using pre-fill reagents in sealed cartridges, ready to use disposables for easy and convenient handling. 100-1000 uL sample volume S/W guide for setup Safe data entry and data management

The LightCycler 2.0 System

LightCycler Training & Application

Viraphong Lulitanond, Ph.D.

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Molecular Automation

LightCycler SystemFeatures
100l / 20l Capillaries Multicolor Detection / Multiplex-PCR

Fast and Accurate Quantification

Flexible Detection Formats Generic Reagents

Mutation & SNP Detection Elimination of Contamination Risk Research & Food Safety Rapid Real-Time PCR

Entering into a new dimension: LightCycler Assay Formats


SYBR Green I Simple Probe Hybridization Probe TaqMan Probe T M P b

Viraphong Lulitanond, Ph.D.

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Molecular Automation

LightCycle Accessories
LightCycler Capillaries: 100l and 20l LightCycler Carousels for 100l and 20l Capillaries

LightCycler Adaptor Cooling Block and Capping Tool

Light Cycler 2.0 Characteristics


Throughput : g p Time : 32 Rxn ~ 30 minutes Detection channel : 6 for multiplex PCR and multicolor mutation analysis capillary tube

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 :

Viraphong Lulitanond, Ph.D.

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Molecular Automation

Light Cycler : IVD kit


HAV Quantification Kit HSV 1/2 Detection Kit EBV Quantification Kit Parvo B19 Quantification Kit MRSA Detection Kit VRE Detection Kit Pseudomonas Kit Staphylococcus Kit Enterococcus Kit Candida Kit Factor V Leiden Kit Factor II (Prothrombin) kit HER2/ neu Quantification Kit Q tifi ti NAT 2 Mutation Detection Kit CYP 2C9 Mutation Detection Kit CYP 2C19 Mutation Detection Kit ApoB Mutation Detection Kit ApoE Mutation Detection Kit CK20 Quantification Kit DPD Quantification Kit TP Quantification Kit TS Quantification Kit t(9;22) Quantification Kit t(14;18) Quantification Kit t(8;21) Quantification Kit t(15;17) Quantification Kit Etc.

Roche Applied Science

PCR Workflow System

Instruments Software Reagents

Speed

Flexibility

Safety

Reliability

Success

Components

Viraphong Lulitanond, Ph.D.

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Molecular Automation

Viraphong Lulitanond, Ph.D.

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