2011 course on molecular diagnostic automation - part 2 - amplification

Patrick MerelBiomedical Innovation Platform (PTIB), Pessac, France

[email protected]

PCR Automation in Molecular Diagnosticpart 2

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From Waterbath to PCR Chips

DNA diagnostic mostly PCR diagnostic

PCR challengers: small market share but robust alternatives

Automation of PCR:

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Automation of PCR setup

Robotic friendlythermocyclers

Thermocyclers evolution

integration into fully automated platforms

microfluidics? PCR chips?

no PCR at all?

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Siemens Branched DNA (bDNA)

NASBA/TMA

Invader

bDNA

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Siemens bDNA latest Automated System

Bayer Versant 4404

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Siemens bDNA Assay Workflow

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bDNA in-house Full Automation

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Digene’s Hybrid Capture Technology

•Same day, objective results using a microplate test format.•High throughput automation for handling increasing test volumes.•Sensitivity comparable to many other target amplification technologies, without the complexity of these other test methods.

•Minimal required specimen preparation, allowing samples to be processed quickly and efficiently.•Protection against cross contamination problems that can occur with some target amplification assays.•Hybrid Capture tests are available to detect human papillomavirus (HPV), chlamydia trachomatis (CT), neisseria gonorrhea (GC), and cytomegalovirus (CMV); the latter is available in tube format only.

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Hybrid Capture Next Generation Platforms

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Signal Amplification with the Invader reaction

In the first reaction, two oligonucleotides - a primary probe and an Invader® oligo - associate with a specific region of DNA to generate a one-base over lapping structure at the nucleotide being interrogated.

IF the variation or sequence in question is present, an overlapping structure is created with the mutant probe and the Invader® oligo on the target. The patented Cleavase® enzymes specifically cleave the primary probes that form overlapping structures with the Invader® oligo, releasing the 5' flaps plus one nucleotide. In the absence of the specific target, no flap is released.

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Released flaps from the primary reaction serve as Invader® oligos in a second, simultaneous invasive cleavage reaction on a labeled, synthetic oligo, the fluorescence resonance energy transfer (FRET) probe. Cleavage of this FRET probe results in the generation of a fluorescent signal.

Because these two cleavage reactions occur simultaneously, they can produce 1 million to 10 million labeled cleavage products per target sequence. A standard 4-hour reaction produces over 10 million-fold signal amplification.

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4hrs incubation

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IVD products ASRsASRs

InPlex CF FII FV

UGT1A1 MTHFR 677 MTHFR 1298

HPV HR-16/18

HPV 16/18 HCVg

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Invader Signal Amplification Evolution

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Invader Plus® methodology

•Utilizes basic PCR amplification

•Coupling PCR with Invader® chemistry brings the best of both worlds

Invader® InPlex™ Technology

•Partnership with 3M, a world leader in health care innovation

•Combines performance, ease of use of Invader® chemistry with 3M microfluidic technology

•Enhances and extends the Invader® chemistry

•Speed to result, Efficiency,Ease of use

•Utilizes existing laboratory equipment

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Target Amplification by TMA

Transcription Mediated Amplification:TMA

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Target Amplification by realtime TMA

Gen-Probe (TMA)๏HIV-1/HCV Procleix assay๏HIV-1/HCV/HBV Procleix Ultrio assay

๏HIV-1, HCV Aptima Qual. assays๏CT/GC Aptima Combo 2๏MTD

Millipore/Gen-Probe (rtTMA)๏Pseudomonas aeruginosa

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Target Amplification by NASBA

Biomerieux-USA๏Nuclisens EasyMag+EasyQ๏Nuclisens HIV-1 QT

Biomerieux-Europe๏Nuclisens EasyMag+EasyQ๏HIV-1, Enterovirus, RSV

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PCR and Diagnostic

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■ 1984 : presented by K. Mullis■ 1988 : 1st PCR by RK. Saiki■ 1991 : 1st TaqMan PCR by PM. Hollan and PCR

patent rights acquired by Roche■ 1992 : Realtime PCR concept by R. Higuchi■ 1996 : Lightcycler launched by Idaho Tech.■ 1997 : LLightcycler launched by Roche■ 2003 : HRM methods introduced by Idaho Tech.■ 2009 : LightCycler1536 introduced by Roche

...in just 25 years

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http://fr.wikipedia.org/wiki/1988
















Automating PCR Operations

Plasticware options

Tubes and plates format

Sealing plates

Instrumentation for regular PCR

robotic friendly?

integration friendly?

Instrumentation for realtime PCR

robotic friendly?

integration friendly?

PCR offering in the IVD market

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Microtiterplate formats for PCR

Various format of 96 well plate format

Up to 384 and 1536 well plate format 20

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Fluidigm, Illumina, LifeTech: New Formats?

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96.96 Dynamic Array IFC

48.770 Digital Array IFC

Fuidigm EP1

or BioMArk

Producing up to 9,216 real-time qPCR reactions in the same amount of time as a single 384-well plate, while using just 1/200th the amount of reagents.

Digital Array

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48-well polypropylene reaction plates

and

Optically clear pressure-sensitive adhesive film

The Eco thermal system incorporates a precisely electroformed hollow silver block that is heated and cooled by a single Peltier device. The hermetically sealed hollow block contains a conductive fluid and two opposing agitators driven by electromagnetic motors. During PCR cycling, these agitators rapidly circulate the fluid throughout the hollow block, transferring heat from the Peltier device evenly across the block. This unique design virtually eliminates thermal non-uniformity and block edge effects, providing a new level of thermal performance of ± 0.1°C well-to-well uniformity across the 48-well plate.

Back to the 48’s

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The OpenArray® Real-Time PCR Instrument and OpenArray AccuFill™ System combination enables the processing of hundreds to thousands of samples per day enabling mid-density, high-throughput results with ease of use. Reliable multiple sample loading— fill 3,072 through-holes (33 nL each) without cross-contamination and with minimal hands-on time. Economical, highly scalable approach—run up to three OpenArray Plates simultaneously on the OpenArray Real-Time PCR Instrument

OpenArray

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AlphaHelix AmpXpress: a very rapid thermal cycler utilizing SuperConvection™ technology that make the instrument significantly faster than conventional thermal cyclers. 40 ycles in 20 minutes.

Compared to conventional thermal cyclers, where thermal homogenization occurs by convection at normal gravity ( 1 x g ) , A m p X p r e s s u t i l i z e s SuperConvection, at 3000 x g, together with extreme heating and cooling, to enable prev ious ly unach ievable temperature ramping rates (measured i n - t ube ! ) and i n s t an t t he rma l homogenization within the samples, that lead to more efficient amplification and time savings.

Innovation in ‘regular’ PCR

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in-tube temperature measurements of reaction temperature

Capillette is a small cartridge containing four chambers that we fill with your reagents for one complete PCR.

Simply add your DNA template to a PCR tube, place Capillette in the top and spin it in a centrifuge to empty the reagents into the tube.

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Icubate: iCubate Cassettes are custom made to

automate molecular protocols such as PCR, ligation, hybridization and detection. Possible applications include pathogen identification from various sample sources.

An iCubate system includes one Reader, one Processor (4 units), and a computer with preinstalled software. The internal and external barcode scanners located throughout the system will ensure error free processing and tracking of your samples.

Propr ietary ARM-PCR technology (Amplicon Rescued Multiplex PCR) allows multiple targets to be amplified in one reaction.

Proven array detection technology analyzes multiple targets at once.

Plug and play operation, no need for extensive training.

Up to twelve units can be linked together to run up to 48 samples simultaneously.


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Icubate:

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Idaho Technologies: FilmArray: The FilmArray integrates

sample preparation, amplif ication, detection, and analysis into one easy to use system capable of massively multiplexed PCR. Fully automated, multiplex PCR capable of detecting greater than 100 targets in one sample in less than one hour.


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Inject unprocessed sample and water, start the run and walk away System integrates sample preparation, amplification,

detection and analysis All necessary reagents are freeze dried in one pouch

and are stable at room temperature

Closed system prevents cross-contaminationMultiplexed testing analyzes up to 120 tests per

sample Rapid results in 1 hour from sample injection

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Automation of PCR procedures

๏Automation of PCR setup

๏Automation through thermocycler integration

๏In house robotics

๏A new era for automated PCR: realtime PCR

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High Throughput PCR wanted

๏Increasing number of tests

๏Increasing number of samples28

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PCR thermocyclers multiple 96 well blocs

http://www.biorad.com

http://www.thermo.com

http://www.appliedbiosystems.com

http://www.biometra.de 29

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http://www.mjresearch.com

http://www.mjresearch.com

http://www.biometra.de

http://www.biometra.de

ü Automation of lid movements

ü Automation of PCR programs

ü Biorad Tetrad

ü AB 9700 AutoLid

ü MWG Primus HT

ü Biometra Trobot

ü ThermoHybaid

Thermocyclers for a complete robotic integration

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Closing the Tubes!

...to pre-cut, thin adhesive backed aluminum foil with a roller as a simple and cost effective system to seal PCR plate...to auto-sealing lids

and heat sealers

From silicone-like sheet with a removable plastic backing...

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MWG-Biotech “no cross contamination” Patented Option

NCC-PCR ensures absolutely airtight sealing of each individual cavity combined with the simplest, touch free operation and compatibility with all current thermal cyclers (0.2ml Block).

In order to avoid contamination through aerosols every cavity in the microplate is closed with individual seals. The opening and closing of the reaction vessels takes place touch-free with the patented "Deckel handler" - no hand contact.

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Thermocyclers and “Low-End” Robotic Integration

Linking a generic workstation with a thermocyler + motorized lid, like here with a BCI Biomek 2000 + Ptc200 from MJR

What is possible:

๏ PCR setup onto the workstation

(oil addition)

๏ manual transfer to the

thermocycler (plate sealing)

๏ or robotic transfer after reaction

setup (oil)

๏ Run the PCR

๏ Transfer the plate back

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1- the DNA+PCR mix is transferred to PCR tubes

2- the primers mixes are then distributed in the PCR tubes

PCR Setup on the Biomek 2000 for generic HLA typing

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Generic HLA-DRB1-DQB1 typing with the Biomek 2000

Two times 40 PCRs (1 PE-9600 plate) are setup for generic HLA-DRB1 - DQB1 typing of t w o p a t i e n t s . T h e P C R reactions are prepared on the Biomek 2000 in 9 minutes.

Four PCR plates (8 patients) can be managed simultaneously by the Biomek 2000. (On the other hand 4 thermocyclers will be needed)

PCR control

Specific PCR

DQB1 PCR

DRB1 PCR

HLA- DRB1*03/07HLA-DQB1*02/02

HLA- DRB1*15/07HLA-DQB1*02/0602

DQB1 PCR

DRB1 PCR

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Going further with Thermocycler Integration

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Thermocycler Modules and Robotic Integration

Integration of a remote PCR block on a MultiProbe robotic workstation from Perkin Elmer

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Thermocyclers and High-End Robotic Integration

epMotion Eppendorf Workstation39

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PSS, Niagala Bio-Station FDX

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Roche Diagnostic Cobas Taqman 96 integration through the Docking Station to the Cobas Amplirep.

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๏Automation of plate transportation

๏Automation of lid movements

๏Automation of PCR programs

๏Biorad Tetrad

Full Robotic Integration

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Automation of plate transportation

Automation of lid movements

Automation of PCR programs

ThermoHybaid, Bio-Rad


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Automation of plate transportation

Automation of lid movements

Automation of PCR setup

Automation of PCR programs

Various robotic arms available


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Plate sealers

Robotic arms or grippers

Accessories for Full Automation

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Velocity11's PlateLoc Thermal Plate Sealer.

As a complementary machine to the PlateLoc, the PlatePierce pierces a wide variety of seals on 96 or 384 well microplates in only 4 seconds of cycle time.

Accessories for Full Automation

Plate sealer

Compatible plates: All SBS standard microplates in 96 and 384 well formatsCompatible seals: A wide variety of both pierceable and peelable seals.

Plate piercer

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Integration of PCR instrumentation

Integration of Beckman Coulter Inc. Biomek FX with thermocyclers (AB-9700, MJR Tetrad…etc) and plate sealer (ABGene).

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LabAutomation 2002 Show Room

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Protedyne BioCube Workstation

The Protedyne PCR BioCube™ is the first unit of its kind that services

up to eight PCR machines(32 blocks) with a single robot.

Materials can enterand exit the PCR BioCube™ via

input and outputconveyors for unattended operation.

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Protedyne BioCube Workstation

The Protedyne PCR BioCube with AB-9700 thermocyclers

Protedyne has also very recently announced the integration of

Roche LC480 thermocycler in the BioCube.

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Fast PCR with integrated detection: the revolution

The Lightcycler integrates a 24 sample micro-volume fluorimeter with an air thermal cycler delivering accurate and high-speed temperature control.

The Lightcycler is the fastest thermal cycler in the world. Thirty cycle reactions can be run in as little as six minutes while an integrated fluorimeter monitors reactions as they occur. The Lightcycler eliminates the need to perform both amplification and separate product analysis allowing unprecedented throughput.

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RealTime PCR Technologies 1 & 2

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Molecular Beacons

RealTime PCR Technologies 3...

TaqMan Probes, Hydrolysis Probes, 5’-Nuclease Process

HRM

Lux Primers

Scorpion Primers

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Realtime PCR Instrument 1

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Company Instrument Capacity; Labware Type of Thermocycler Robotic Friendly(Yes, No, Not

Specified)

Web Address

AB/Life Technologies

StepOne, StepOne+ 48, 96 fast block N

appliedbiosystems.comAB/Life Technologies

7500 96 well plate Peltier based, fast block option N

appliedbiosystems.comAB/Life Technologies7900HT 96, 384 well plate, or

TaqMan arrayPeltier based, fast

block option Yappliedbiosystems.comAB/Life Technologies

ViiA 7 96, 384 well plate, or TaqMan array fast block Y

appliedbiosystems.com

BD BD Max24 individually

controlled chambers; microfluidic cartridge

fast cycler

Y; Integration of sample preparation,

amplification and detection

bd.com

Bioer Technology Line-Gene,2,K 33,66,48 tubes Sandwich thermal-electronic Peltier N bioer.com.cn/en

Biogene Insyte 96 well plate fast cycler NS biogene.com

Bioneer Exicycler 96 well plate Peltier based NS bioneer.com

Bio-rad MiniOpticon, MyIQ2, CFX89, CFX384 48,96,96,384 well plate Peltier based N bio-rad.com

Cepheid

SmartCycler 16 independent units; proprietary tubes fast cycler N

cepheid.comCepheid GeneXpert1, 4, 16 independent

units; proprietary cartridge

fast cycler

Y; Integration of sample preparation,

amplification and detection

cepheid.comCepheid

GeneXpert Infinity 48 independent units; proprietary cartridge

Continuous flow fast cycler Y

cepheid.com

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Realtime PCR Instrument 2

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Company Instrument Capacity; Labware Type of Thermocycler

Robotic Friendly(Yes, Not, Not

Specified)

Web Address

EppendorfMastercyler ep

realplex96 well plate Peltier based N eppendorfna.com

Evogen Evocycler HD12 12 tubes-proprietary fast cycler N evogen.com

Focus 3M integrated Cycler 96 well disc fast cycler N integratedcycler.com

GeneSystems GeneDisc Cycler6 samples-6 tests;

CD formatfast cycler N genesystems.fr

Illumina Eco Real-Time 48 well platePeltier based

N illumina.com

Qiagen Rotor-Gene Q36 tubes; 72, 100 proprietary tubes

fast cyclerN qiagen.com

Roche Diagnostics

LightCyler V1, V2 32 glass capillaries;fast cycler

Nroche-applied-science.com

Roche Diagnostics

LightCyler 480; 153696, 384 ; 1536 well

plate

fast cyclerY

roche-applied-science.com

Roche DiagnosticsCobas Taqman 48

2x24 proprietary tubes

Peltier basedN

roche-applied-science.com

Roche DiagnosticsCobas Taqman 48

2x24 proprietary tubes

Peltier basedN

roche.com

Roche Diagnostics

Cobas Taqman 964x24 proprietary

tubes

Peltier based

Yroche.com

Stratagene-Agilent Technologies

Mx3000, Mx3005P 96 well plate Peltier based N stratagene.com

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AlphaHelix QuanTyper Superconvection and Capilette

Applied Biosystems/Life TechnologiesViiA 7 Realtime PCR with 96, 384 well plate and Taqman Array microfluidic cards

OpenArray with 3072 through hole plate

Fuidigm digital PCRBioMark Realtime PCR with 48x48 (2304) or 96x96 (9216) Dynamic, Digital and Access Array IFCs

Roche Applied SciencesLightCycler 1536 Fast Realtime PCR

Small initiatives like

GeneSystems’s GeneDisc realtime PCR cycler

Enigma Diagnostics

Innovation in realtime PCR

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AlphaHelix QuanTyper 48: the first qPCR instrument to utilize SuperConvection, making it the world's most rapid qPCR instrument.

40 cycle qPCR including melt analysis in 15 minutes

48 samples per run

Quadruplex

20 - 200 µl volume range

Rapid and unique HRM analysis

AlphaHelix SuperConvection realtime PCR

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The rotating samples are heated using a circular high-energy IR source.

Capillette is a small cartridge containing four chambers that we fill with your reagents for one complete PCR.

Simply add your DNA template to a PCR tube, place Capillette in the top and spin it in a centrifuge to empty the reagents into the tube.

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LifeTechnologies Open Array realtime PCR

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The OpenArray® Real-Time PCR Instrument and OpenArray AccuFill™ System combination enables the processing of hundreds to thousands of samples per day enabling mid-density, high-throughput results with ease of use. Reliable multiple sample loading— fill 3,072 through-holes (33 nL each) without cross-contamination and with minimal hands-on time. Economical, highly scalable approach—run up to three OpenArray Plates simultaneously on the OpenArray Real-Time PCR Instrument

OpenArray

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Fluidigm Digital Array realtime PCR

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96.96 Dynamic Array IFC BioMArk reader

Producing up to 9,216 real-time qPCR reactions in the same amount of time as a single 384-well plate, while using just 1/200th the amount of reagents.

Digital Array

IFC Controller HX

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Roche LC-1536 realtime PCR

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•Increase throughput fourfold

•Generate 1,536 data points in a single run in less than 50 min.

•Use significantly less reagent and sample with reaction vol. of just 0.5-2.0 µl.

1536 ‘regular’ realtime PCR

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GeneSystems GeneDisc Cycler

The GeneDisc uses a hybrid technology between the DNA chip and real-time PCR microplates that proves to be especially relevant for routine diagnostic applications.

It is a single-use micro-laboratory of the size of a compact disc.Its rim is engraved with 36 reaction microchambers preloaded with the reagents necessary for detecting and quantifying the required targets.

Wells at its centre will be filled with the purified bacterial DNA.A fine network of microchannels automatically and uniformly transfers the bacterial DNA samples to the reaction chambers.

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GeneSystems GeneDisc

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PCR Instrumentation

So What About Automation?

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Popular 96 Well Plate Instrumentation

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Difficult to Automate

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Fast PCR Popular Instrumentation

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Automation with Capillaries?

The “capillary” concern

The “carousel” concern68

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๏30 µL x 100-wells: GeneDisc 100 (heat-sealed plate)๏0.1 mL x 72-wells: GeneDisc 72 (heat-sealed plate)๏0.1 mL x 72-wells: 0.1 mL strip tubes (strips of caps)๏0.2 mL x 36-wells: 0.2 mL tube (attached cap)

Rotor-Gene Q

Fast PCR Instrument: Corbett Researchnow part of Qiagen

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PCR Instrumentation

Solutions for Realtime PCR Automation?

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QPCR robotic for 96-384 sample batch

The ABI PRISM® 7900HT Sequence Detection System is a high-throughput real-time PCR system.

• An Automation Accessory combined with 384-well plate capability make the 7900HT system ideally suited to meet the high-throughput requirements.

• Key applications include gene expression quantitation and the detection of single nucleotide polymorphisms (SNPs) using the fluorogenic 5’ nuclease assay.

Key Features and Benefits

• Interchangeable formats provide improved throughput and flexibility

• Custom Automation Accessory provides 24-hour unattended operation

• Hand-held and integrated bar code readers simplify sample tracking

• Continuous wavelength detection from 500-660 nm allows the use of multiple fluorophores in a single reaction

Applied BioSystems PRISM® 7900HT

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QPCR Robotic Integration

High Throughput Core Systems already exist for PCR automation

Combination of BCI core system + Prism 7900 allows configurations for:

NA extraction

PCR setup

Plate sealing

Prism 7900 loading

Up to 384 PCR every 2.5hrs72

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High Throughput Robotic QPCR with multiple formats

The Roche LighCycler 480

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New Step in High Throughput and rapid QPCR

The LightCycler 480

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LightCycler 480 and Caliper’s Twister II

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Roche LC480 and Automation

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Robotic QPCR Process

LC480 and Caliper LS

LC480 and Protedyne Radius

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Biomek Nx-Sp8 / LC480 integration

The players: Beckman Coulter, Roche Diagnostics, AIG

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An integration from The Biomedical Innovation Platform @ University Hospital of Bordeaux, France with the help of AIG, Germany.

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Very High Throughput QPCR with the LightCycler 1536

•Increase throughput fourfold.•Generate 1,536 data points in a single run in less than 50 minutes.•Use significantly less reagent and sample with reaction volumes of just 0.5-2.0 µl.

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PCR Instrumentation

Automation of unconventional realtime PCR formats?

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Automation of PCR Setup with the MagnaPure LCs

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MagnaPure LC2 MagnaPure LC

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Automation of PCR Setup with the QIAgility

The QIAgility uses a single-channel pipet to automate PCR setup - up to 96 PCR reactions can be set up in approximately 30 minutes.

Liquid-level sensing, facilitated by conductive filtered tips, enables high-precision pipetting

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Automated PCR setup in all formats

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Integration of PCR Setup with the QIAsymphony AS

The QIAsymphony AS extends the capabilities of the QIAsymphony SP to include automated assay setup.

The new module interfaces with the QIAsymphony SP and is operated via the same easy-to-use, intuitive software.

For integrated operation, samples processed on the QIAsymphony SP can be transferred to the QIAsymphony AS, reducing manual handling steps and documentation.

The QIAsymphony AS provides active cooling of reagents, eluates, and assays, enabling safe and reproducible assay setup.

The QIAsymphony AS enables setup of multiple assays per run or sample and supports artus® products and other QIAGEN products for PCR.

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Automated PCR setup in all formats

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Robotic Friendly QPCR Instruments

The Parallab 350 (BioGene) is an

automated nano pipetter, including a magnetic bead

station/separator and a fast thermocycler

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PCR Instrumentation

Automation Through Integration

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Full Integration with the Cepheid GeneXpert

The GeneXpert cartridge-based PCR detection system integrates automated sample preparation with real-time PCR analysis. It can handle up to 5 mL of aqueous sample such as medical or environmental swab extracts, bioaerosols, or serum. Dry on-board reagents, easy sample handling and setup, and a totally internally controlled reagent system enable true random access performance with results available in 30 minutes or less.

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Cepheid GeneXpert

The GeneXpert System is a 1 to 4-site, random access instrument integrating real-time amplification and detection features seen in the SmartCycler System, but delivering results from unprocessed samples in less than 30 minutes.

The internal module is the common technology link between the SmartCycler and GeneXpert, performing real-time amplification and detection.

The GeneXpert automates sample preparation, integrating the complex steps of DNA extraction in the microfluidic cartridges.

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Cepheid GeneXpert Family-1 to 16 units

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A New Step in MDx Automation

Cepheid Infinity / AACC 2008Up to 2,074 test results in a mere 24 hour period. Less than 52 minutes time to first result. Less than 2 minutes sample prep time per test. MRSA, EV, GBS tests available.

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Iquum Lab-in-a-Tube Platform

Liat Analyzer:The Liat system, consisting of the Liat Analyzer and disposable Liat Tubes, automates all nucleic acid testing processes, including reagent preparation, target enrichment, inhibitor removal, nucleic acid extraction, amplification and real-time detection, in one portable device.

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Iquum Lab-in-a-Tube Platforms

The Liat FlowCycler can amplify 48 samples in parallel and is expandable to process 96 or more samples.

The system has 2 bays, each capable of accepting up to 3 Liat™ Tube Strips. Each Liat Tube Strip comprises 8 flexible PCR tubes for a system throughput of 48 samples.

The spacing of the individual flexible tubes of the Liat Tube Strip matches that of 8-well PCR tube strips or 8-well columns of a 96 well plate, thus readily allowing integration of the FlowCycler system into existing liquid handling system.

For example, a 50μl rapid PCR can be accomplished in

as little as 7 min for 30 cycles.

Liat FlowCycler

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Microfluidic based fully integrated platform

A microfluidic real time nucleic acid analyzer, the BD-Max by HandyLab.

This system is capable of processing raw patient samples, blood, urine, swab, CSF, etc. to a result in about one hour.

It's PCR Real Time format will accept FDA cleared, analyte specific reagents (ASR) and/or your homebrew assays.

HandyLab's proprietary unitized reagents and microfluidics cartridge radically reduce the chance of a contamination event in your laboratory.

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PCR Instrumentation

Automation Through IVD Industry Offering

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Numerous molecular diagnostic kits providers on the market with partial offer: Qiagen (NA extraction + reagents), Nanogen (reagents), Italian Market, Eastern Europe, Korea, China, Taiwan...etc. See various scenario at the end of the course.

•Realtime based assay developments•Instruments availability

•Industry complete offering instrument+reagents•Recent increase in rtPCR IVD assays

•Abbott Diagnostics•m2000sp+m2000rt•HIV, HBV, HCV, CMV, EBV, (HSV, VZV), CT/NG

•Roche Diagnostics•Cobas Ampliprep + Taqman 48, 96•HIV, HBV, HCV•MagnaPureLC2 +LC2?•MagnaPure96+LC480?•CMV, EBV, HSV, VZV•upcoming Cobas 6800

•Cepheid•GeneExpert, Infinity•EV, MRSA, GBS

•Biomerieux•EasyMag+EasyQ•HIV, Enterovirus, RSV

•BD•BD-Max; GBS,NG•Viper SP; CT/GC

Realtime PCR: instruments+reagents offers

•Gen-Probe/Millipore•Milliprobe•contamination detec.•upcoming Panther

•Qiagen•QIAsymphony SP+AS + Rotor-Gene Q?•upcoming QIAensemble

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•Siemens•Versant kPCR•HIV, HBV, HCV, CT/GC

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Abbott Molecular PCR automation

Ct/Ng Screening

HIVViral Load

HCVViral Load

CMVViral Load

HBVViral Load

Ct Screening

Open modes

VIROSEQGénotypage

M200RTPrism 7000

HSV

M200RT

VZV

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Data

Sample Prep(m2000 sp V3.0)

LIS

(m2000rt)

Router

(m2000 rt V2.1)

Abbott Lab organization solution

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Continuous Flow Realtime PCR

๏1st option:

Automatic extraction on the Cobas Ampliprep + realtime PCR on the Cobas Taqman 48

๏2nd option:

Automatic extraction + Automatic realtime PCR on the Cobas Ampliprep+Dock+Taqman 96

๏200 sample/day

๏HCV, HIV, HBV

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Roche COBAS TaqMan 96

Thermocyclers

Sample racks K-carriers waiting for TC

Robotic arm

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Roche Cobas Docking Station

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Siemens Molecular - Versant K-PCR

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Realtime PCR automation: the new dilemma

Major Diagnostic companies reagents

Molecular biology companies reagents

Molecular Diagnostic companies reagents

High cost (40-50 €)Reagents rental programsLow cost of instruments

Lower cost (20-30 €)QC, QA, standards

providedInstruments not provided

Unexpensive (<10 €)QC, QA, standards to

performInstruments not provided

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Roche Diagnostic Lab. Setup proposition

HIV 9284HCV 5552HBV 1009CMV 10313EBV 5104HSV 2749VZV 1280Adenovirus 1 428

HHV6 Quantitatif 1 406

VRS 953

Grippe 894

Enterovirus 725

HHV8 104

BK 100

Parvovirus B19 100

Norovirus 100

SARS, Coronavirus 50

Grippe Aviaire H5N1 50

ParaInfluenzae 608

Metapneumovirus 300

42109

CE-IVD

HomeBrew

CMVEBVHSVVZV

HIVHBVHCV

+ HomeBrew

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Abbott Molecular Lab. Setup proposition

Données

Sample Prep(m2000 sp V3.0)

LIS

(m2000rt)

Router

(m2000 rt V2.1)

Evo 75 Nat HSVVZV

HIVHBVHCVCMVEBV

CE-IVD

HIV 9284HCV 5552HBV 1009CMV 10313EBV 5104HSV 2749VZV 1280

35291

CE-IVD

102

102

courtesy of A Esnault 2008 Abbott

9:00 11:0010:00 12:00 14:0013:00 15:00 17:0016:00 18:00

Lundi

Mardi

Mercredi

Jeudi

Vendredi

Lab. Setup with M 2000 sp V3.0 et EVO 75

Extraction HIV 48 tests PCR HIV 48 tests Sp1 rt1

Sp2 rt2Extraction HCV 48 tests PCR HCV 48 tests Sp1 Extraction CMV 96 tests rt1 PCR CMV 96 tests

PCR HBV 24 tests Sp2 rt2Extraction HBV 24 tests



Sp2 rt2Extraction HCV 48 tests PCR HCV 48 tests

Sp1 Extraction CMV 96 tests rt1 PCR CMV 96 tests



Sp2 rt2Extraction HCV 48 tests PCR HCV 48 tests

Sp1 extraction EBV 48 tests rt1 PCR EBV 48 tests

Sp1 extraction EBV 48 tests rt1 PCR EBV 48 tests

ABI PCR VZV 24 testsEVO 75 Ext VZV 24 tests

ABI PCR HSV 24 testsEVO 75 Ext HSV 24 tests

103

103

Beyond HIV,HBV,HCV-CMV,EBV,HSV,VZVMost comprehensive additional menu in infectious disease:

1) Nanogen-Elitech 2) Qiagen

AdenovirusHHV6VRSFluEnterovirusHHV8BK

FluEnterovirusParvovirus B19SARS, CoronavirusH5N1

AB Prism compatible/CE-IVD AB Prism compatible/CE-IVDH5N1 Roche LC compatible

Adenovirus 1 428

HHV6 Quantitatif 1 406

VRS 953

Grippe 894

Enterovirus 725

HHV8 104

BK 100

Parvovirus B19 100

Norovirus Cepheid

SARS, Coronavirus 50

Grippe Aviaire H5N1 50

ParaInfluenzae diagenode

Metapneumovirus diagenode

5810

CE-IVD

104

104

Realtime PCR Market Evolution

Major Diagnostic companies

Molecular Diagnostic reagents companies

- Main molecular markers(HIV, HBV, HCV, CT/GC)- CE-IVD, FDA approved

- Instruments

-Numerous minor markers-Rarely major markers

-ASR, RUO-Rarely instruments

Recentlymajor diagnostic companiesenhanced menu for minor

makers

-Full menu-CE-IVD

-Instruments

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105

MDX, PCR, Automation in conclusion

✓PCR diagnostic switched from PCR to realtime PCR✓but still options for high-tech automated regular PCR+Arrays instruments

✓Realtime PCR switched to Fast PCR✓from 96 well plates to capillaries to 96, 384 and 1536 well plates

✓Realtime MDx assays on 2 instruments only✓NA extraction/PCR setup + Realtime PCR

✓in house or MDx industry numerous solutions

✓Integration of realtime MDx assays on a single instrument or expandable/connected instruments✓Roche Cobas, Qiagen QIAsymphony, Gen-Probe Panther instruments

✓evolution through microfluidic instrumentation (Cepheid, BD/HandyLab..)

106

✓Realtime PCR arrays and gene expression

106

107

107

2011 course on molecular diagnostic automation - part 2 - amplification

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