molecular automation

Molecular Automation

Viraphong Lulitanond, Ph.D. 1

Molecular Automation450 444 Autoanalysers in Clinical Laboratory

Viraphong Lulitanond, [email protected]@

Research and Diagnostic Center for Emerging Infectious Diseases

WhWhyMolecular techniques ?



The Better Choices

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

t itt it

Nucleic acid testing utility in Nucleic acid testing utility in laboratory practicelaboratory practice

blood bank screeningblood bank screening

genetics and cancergenetics and cancer

opportunityopportunity

infectious diseasesinfectious diseases

gg

timetime



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,HIV-1RNA

Ab to HIV-1

10 20 30 40 50 60 70 80 900

Ag or AbLevel

p24 Ag

Days post infection

• Antibodies are detectable approximately 80 days after

Narrowing The Window On Transfusion-Associated HCV Infection

Viral RNA HCV RNA

• Antibodies are detectable approximately 80 days after infection.

• Viral RNA is detectable approximately 23 days after infection.

10 20 30 40 50 60 70 80 900

Days post infection

Viral RNAor AbLevel

HCV RNA

Ab to HCVAb to HCV



Advantage of Nucleic Acid Amplification Techniques

• Less amounts of samplesLess amounts of samples• Sensitivity• Specificity• Rapid• Simple• Offer - Diagnosis (Qualitative)

- Prognosis (Quantitative)- Therapeutic Monitoring (Quantitative)

PCR…In the past…

1998



PCR Process1. Sample preparation (Pre-PCR)2. Amplification (PCR)3. PCR products detection (Post-PCR)

Gel electrophoresisHybridize with specific probe Plate-based hybridization assayy ySequencing

THERMUS AQUATICUS FROM YELLOWSTONE NATIONAL PARK

Source of Taq DNA polymerase



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

blottinggel electrophoresisgel electrophoresis

• Narrow dynamic range• Open tube format (in-house)• Easy false contamination (in-house)



ETHIDIUM BROMIDE STAINING UNDER UV ILLUMINATION

M l l i h kMolecular weight marker

Programmable Temperature Cycler (PCR machine)



Roche Molecular Diagnostics

1998

Roche Molecular Diagnostics

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COBAS AMPLICOR Test ProfileCOBAS AMPLICOR Test Profile•• HIVHIV--1 1 RNA (viral load) RNA (viral load)

HCV RNA( i l l d)HCV RNA( i l l d)

•• HIVHIV--1 1 proviral DNAproviral DNA

HPVHPV•• HCV RNA(viral load) HCV RNA(viral load)

•• HBV DNA(viral load) HBV DNA(viral load)

•• CMV DNA(viral load) CMV DNA(viral load)

•• AmpliScreen HIVAmpliScreen HIV

•• AmpliScreen HCVAmpliScreen HCV

•• HPVHPV

•• HCV qualitativeHCV qualitative

•• HCV genotypingHCV genotyping

•• CC. . trachomatistrachomatis

•• NN. . gonorrhoeaegonorrhoeae

•• AmpliScreen HBVAmpliScreen HBV

•• CYP CYP 450450

•• MM. . tuberculosistuberculosis

•• Beta thalassemiaBeta thalassemia

COBAS TAQMANIntegrated

Evolution of Roche Diagnostic Platforms

Deg

ree

of A

utom

atio

n

COBAS AMPLIPREP

AutomatedSample

Preparation

Walk-away System

AMPLILINKCOBAS

AMPLICORMONITOR

1992 1995 1996 2001 2003MWP

COBASAMPLICOR



Semi-automation systemAMPLICOR assay•Launch in 1992•First ready to use•Standradized kit

TC 9600TC 9600

COBAS AMPLICORCOBAS AMPLICOR

• Launch in 1995• First fully automated amplification and detection• Second generation assays, both qualitative and quantitative assays



COBAS AMPLICORPhotometer

Computer Printer

COBAS AMPLICOR SystemAmplification and Detection

• Load samples, run information and reagents, start COBASAMPLICOR and walkaway• Parallel mode: load 2nd set of specimens after amplification of 1st• Parallel mode: load 2nd set of specimens after amplification of 1st

Thermal cycler B

Pipettor Photometer

Incubator

Thermal cycler A

Detection positions

Reagent racks D-cups Wash station



COBAS AmpliPrepAutomation Sample Preparation

Mi i i• Minimum instrument set up• Full automation• Flexible• Continuous operation:

Additional samples• Onboard storage of prep.

samplessamples• 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



Real Time PCRhSimultaneous amplification and detection ( no separate detection )hKinetic quantification in the log phase of the reactionhData is collected at each cycle and are displayed immediatelyy p y y

hQualitative and / or Quantitative analysishMutation Analysis / SNP hReduce assay time

I th h t

Common Features and Benefits

hIncrease throughputhBroad dynamic rangehClose tube format hEliminate contamination risk

Monitoring of PCR Reactions

Y = X(1+E)n



PCR and the Problem of Quantification

high concentration /high efficiencyloglog--phase analysisphase analysis endend--point analysispoint analysishigh concentration /low efficiencylow concentration /high efficiency

N : number of amplified moleculesn : number of amplification cycles

N

p yp y

Main problems of PCR:

n

Main problems of PCR:1. Plateau effect2. Efficiency of amplification

Real-time PCR Detection Formats

SYBR Green I Hybridization Probe Format Hydrolysis Probe Format

Elongation Phase Annealing Phase Elongation Phase



SYBR Green l Format Overview

D t ti D bl t d• Denaturation: Double strand 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 LightCyclerusing SYBR Green I

H2O

1E+1

1E+2

1E+3

1E+4

1E+5

1E+6

1E+7

1E+8

1E 9

Fluorescence -

d (F1) / dT

Fluorescence (

F1)

Template: Plasmid; Target: TNFα; Detection Format: SYBR Green I

1E+9

Cycle Number Temperature °C



Hybridization Probes Format Hybridization Probes Format OverviewOverview

FluoresceinFluorescein

LC LC RedRed••Denaturation:Denaturation: Double strand DNA to Single strand DNA by heat. No FRETFRET

•• Probe hybridization :Probe hybridization : two oligo probe hybridize to DNA strand. Fluorescent signal can be monitored

transfertransferexciteexcite emissionemission

•• Elongation :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



Hydrolysis Probe Format Overview

reporter quencher

COBAS TaqMan 48



COBAS TaqMan 48

Size (45 x 75 x 50) cm

Weight ∼ 53kg

Batch Size 2 x 24 (50 – 100 μl)

Channels 4 (520 nm, 575 nm, 645 nm, 725 nm)

Disposable Plastic tube (k-tube)

modified k-tray

Automation Manual transfer required

COBAS TaqMan AnalyzersCommon Features and Benefits

Feature• Kinetic PCR

Benefit• Broad dynamic range• No separate detection

• Integrated Quantification Standard requires no external standard curves

• Closed Tube Format• Four color channels

• No separate detection• Assay time reduction

• Increases Throughput• Provides internal control

• Eliminates contamination risk• Allows multiplexing• Will allow additional formats

• Sample vessel: k-tube or k-plate

• Will allow additional formats (Hybridization Probes)

• Easy handling• Automation through AmpliPrep



COBAS TaqMan Analyzer Thermal cyclers

TC lid

TC cover

Two independent thermal cyclers for maximum flexibility

COBAS TaqMan 482 Applications

• IVD (HIV, HCV, HBV, HPV, HSV, CT)

• Utility Channel (in house)



• Two independent Thermal Cyclers– two different protocols simultaneously

Cobas TaqMan 48 - Utility Channel

– 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• Customer can use system for own applications

Total PCR Automation

COBAS AMPLICOR

COBAS TaqMan 48

COBAS TaqMan 96



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

•True walk - away automation for l i id ifi i

MagNA Pure LC System

nucleic acid purification

• 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• Broad variety of sample materials (blood, plasma, serum, sputum, tissue, feace, etc.)



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



LightCycler System…Features

Fast and Accurate Quantification

Mutation & SNP Detection

Flexible Detection Formats

100μl / 20μl Capillaries Multicolor Detection / Multiplex-PCR

Generic Reagents

Rapid Real-Time PCR

Elimination of Contamination RiskResearch & Food Safety

Entering into a new dimension: LightCycler Assay Formats

SYBR Green I

Simple Probe

Hybridization Probe

T M P bTaqMan Probe



LightCycle AccessoriesLightCycler Capillaries:

100μl and

LightCycler Carousels for100μl and 20μl Capillaries

20μl

LightCycler Adaptor Cooling Block and Capping Tool

Throughput : 32 Rxn

Light Cycler 2.0 Characteristics

g p

Time : ~ 30 minutes Detection channel : 6 for multiplex PCR and multicolor mutation analysis

Disposable : capillary tube

Reaction volume : 20, 100 uL

S ft Li ht C l ft f l i d i t t tiSoftware : Light Cycler software for analysis and interpretation of realtime quantification and melting curve data

Utility : IVD kit and research application



Light Cycler : IVD kit• HAV Quantification Kit• HSV 1/2 Detection Kit

• Factor V Leiden Kit• Factor II (Prothrombin) kit

HER2/ Q tifi ti Kit• EBV Quantification Kit• Parvo B19 Quantification Kit• MRSA Detection Kit• VRE Detection Kit• Pseudomonas Kit• Staphylococcus Kit• Enterococcus Kit

• HER2/ neu Quantification Kit• 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

• Candida KitTP 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 ComponentsSuccess

molecular automation

Documents