2011 course on Molecular Diagnostic Automation - Part 2 - Amplification. This is from early 2011. Prices and Specifications of instruments may have changed. Part 2 of 3

1. Patrick Merel
Biomedical Innovation Platform (PTIB), Pessac, France
pmerel@mac.com
PCR Automation in Molecular Diagnostic
part 2
1
1

2. From Waterbath to PCR Chips
DNA diagnostic mostly PCR diagnostic
PCR challengers: small market share but robust alternatives
Automation of PCR: Automation of PCR setup
Robotic friendly
thermocyclers
Thermocyclers evolution
integration into fully automated platforms
microfluidics? PCR chips?
no PCR at all?
2
2

3. Siemens Branched DNA (bDNA)
bDNA
Invader
NASBA/TMA
3
3

4. Siemens bDNA latest Automated System
Bayer Versant 440
4
4

5. Siemens bDNA Assay Workflow
5
5

6. bDNA in-house Full Automation
6
6

7. 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.
7
7

8. Digene’s Hybrid Capture Technology
8
8

9. Digene’s Hybrid Capture Technology
9
9

10. Hybrid Capture Next Generation Platforms
10
10

11. Signal Amplification with the Invader reaction
IF the variation or sequence in question is present, an
In the first reaction, two oligonucleotides
overlapping structure is created with the mutant probe
- a primary probe and an Invader® oligo -
and the Invader® oligo on the target. The patented
associate with a specific region of DNA to
Cleavase® enzymes specifically cleave the primary probes
generate a one-base overlapping
that form overlapping structures with the Invader® oligo,
structure at the nucleotide being
releasing the 5' flaps plus one nucleotide. In the absence
interrogated.
of the specific target, no flap is released.
11
11

12. Signal Amplification with the Invader reaction
Released flaps from the primary reaction B e c a u s e t h e s e t w o c l e ava g e
serve as Invader® oligos in a second, reactions occur simultaneously,
simultaneous invasive cleavage reaction on they can produce 1 million to 10
a labeled, synthetic oligo, the fluorescence million labeled cleavage products
resonance energy transfer (FRET) probe. per target sequence. A standard 4-
Cleavage of this FRET probe results in the hour reaction produces over 10
generation of a fluorescent signal. million-fold signal amplification.
12
12

13. Signal Amplification with the Invader reaction
IVD products ASRs
InPlex CF FII FV
UGT1A1 MTHFR 677 MTHFR 1298
HPV
HPV 16/18 HCVg
HR-16/18
4hrs incubation
13
13

14. Invader Signal Amplification Evolution
Invader Plus® methodology Invader® InPlex™ Technology
•Utilizes basic PCR amplification •Partnership with 3M, a world leader in health care innovation
•Coupling PCR with Invader® chemistry •Combines performance, ease of use of Invader® chemistry
brings the best of both worlds with 3M microfluidic technology
•Enhances and extends the Invader® chemistry
•Speed to result, Efficiency,Ease of use
•Utilizes existing laboratory equipment
14
14

15. Target Amplification by TMA
Transcription Mediated Amplification:
TMA
15
15

16. 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
16
16

17. Target Amplification by NASBA
Biomerieux-USA
๏Nuclisens EasyMag+EasyQ
๏Nuclisens HIV-1 QT
Biomerieux-Europe
๏Nuclisens EasyMag+EasyQ
๏HIV-1, Enterovirus, RSV
17
17

18. PCR and Diagnostic
...in just 25 years
■ 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
18
18

19. 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
19
19

20. Microtiterplate formats for PCR
Various format of 96 well plate format
Up to 384 and 1536 well plate format 20
20

21. Fluidigm, Illumina, LifeTech: New Formats?
Fuidigm EP1
or BioMArk
96.96 Dynamic Array IFC
48.770 Digital Array IFC
Digital Array
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.
21
21

22. Fluidigm, Illumina, LifeTech: New Formats?
48-well
polypropylene
reaction
plates
and
Optically clear
pressure-
sensitive
adhesive film
Back to the 48’s
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.
22
22

23. Fluidigm, Illumina, LifeTech: New Formats?
OpenArray
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
23
23

24. Innovation in ‘regular’ PCR
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 in-tube temperature measurements
occurs by convection at normal gravity of reaction temperature
(1 x g), AmpXpress utilizes
SuperConvection, at 3000 x g, together
with extreme heating and cooling, to
enable previously unachievable Capillette is a small cartridge
temperature ramping rates (measured containing four chambers that we
fill with your reagents for one
in-tube!) and instant thermal
complete PCR.
homogenization within the samples,
that lead to more efficient amplification Simply add your DNA template to
and time savings. a PCR tube, place Capillette in
the top and spin it in a centrifuge
to empty the reagents into the
tube.
24
24

25. Innovation in ‘regular’ PCR
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.
Proprietary 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 Icubate:
extensive training.
Up to twelve units can be linked together
to run up to 48 samples simultaneously.
25
25

26. Innovation in ‘regular’ PCR
Idaho Technologies:
FilmArray: The FilmArray integrates
sample preparation, amplification,
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.
Inject unprocessed sample and water, start the run Closed system prevents cross-contamination
and walk away Multiplexed testing analyzes up to 120 tests per
System integrates sample preparation, amplification, sample
detection and analysis Rapid results in 1 hour from sample injection
All necessary reagents are freeze dried in one pouch
and are stable at room temperature
26
26

27. Automation of PCR procedures
๏Automation of PCR setup
๏Automation through thermocycler integration
๏In house robotics
๏A new era for automated PCR: realtime PCR
27
27

28. High Throughput PCR wanted
๏Increasing number of tests
๏Increasing number of samples 28
28

29. PCR thermocyclers multiple 96 well blocs
http://www.biorad.com
http://www.thermo.com
http://www.appliedbiosystems.com
http://www.biometra.de 29
29

30. Thermocyclers for a complete robotic integration
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
30
30

31. Closing the Tubes!
From silicone-like sheet
with a removable
plastic backing...
...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
31
31

32. 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.
32
32

33. 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
33
33

34. PCR Setup on the Biomek 2000 for generic HLA typing
1- the DNA+PCR mix is
transferred to PCR
tubes
2- the primers mixes are
then distributed in the
PCR tubes
34
34

35. Generic HLA-DRB1-DQB1 typing with the Biomek 2000
DRB1 PCR
Two times 40 PCRs (1 PE-9600
PCR control
plate) are setup for generic
HLA-DRB1 - DQB1 typing of Specific PCR
two patients. The PCR
reactions are prepared on the DQB1 PCR
Biomek 2000 in 9 minutes.
HLA- DRB1*03/07
Four PCR plates (8 patients) can be HLA-DQB1*02/02
managed simultaneously by the DRB1 PCR
Biomek 2000. (On the other hand 4
thermocyclers will be needed)
DQB1 PCR
HLA- DRB1*15/07
HLA-DQB1*02/0602
35
35

36. Going further with Thermocycler Integration
36
36

37. Automation of PCR procedures
๏Automation of PCR setup
๏Automation through thermocycler integration
๏In house robotics
๏A new era for automated PCR: realtime PCR
37
37

38. Thermocycler Modules and Robotic Integration
Integration of a remote PCR block on a MultiProbe robotic workstation
from Perkin Elmer
38
38

39. Thermocyclers and High-End Robotic Integration
epMotion Eppendorf Workstation
39
39

40. Thermocyclers and High-End Robotic Integration
PSS, Niagala Bio-Station FDX
40
40

41. Thermocyclers and High-End Robotic Integration
Roche Diagnostic Cobas Taqman
96 integration through the Docking
Station to the Cobas Amplirep.
41
41

42. Automation of PCR procedures
๏Automation of PCR setup
๏Automation through thermocycler integration
๏In house robotics
๏A new era for automated PCR: realtime PCR
42
42

43. Full Robotic Integration
๏Automation of plate transportation
๏Automation of lid movements
๏Automation of PCR programs
๏Biorad Tetrad
43
43

44. Full Robotic Integration
Automation of plate transportation
Automation of lid movements
Automation of PCR programs
ThermoHybaid, Bio-Rad
44
44

45. Full Robotic Integration
Automation of plate transportation
Automation of lid movements
Automation of PCR setup
Automation of PCR programs
Various robotic arms available
45
45

46. Accessories for Full Automation
Plate sealers
Robotic arms or grippers
46
46

47. Accessories for Full Automation
Plate sealer
Plate piercer
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.
Compatible plates: All SBS standard
microplates in 96 and 384 well formats
Compatible seals: A wide variety of both
pierceable and peelable seals. 47
47

48. Integration of PCR instrumentation
Integration of Beckman Coulter Inc. Biomek FX
with thermocyclers (AB-9700, MJR
Tetrad…etc) and plate sealer (ABGene).
48
48

49. LabAutomation 2002 Show Room
49
49

50. 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 enter
and exit the PCR BioCube™ via
input and output
conveyors for unattended operation.
50
50

51. Protedyne BioCube Workstation
Protedyne has also very recently The Protedyne PCR BioCube
announced the integration of with AB-9700 thermocyclers
Roche LC480 thermocycler in the
BioCube.
51
51

52. Automation of PCR procedures
๏Automation of PCR setup
๏Automation through thermocycler integration
๏In house robotics
๏A new era for automated PCR: realtime PCR
52
52

53. 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.
53
53

54. RealTime PCR Technologies 1 & 2
54
54

55. RealTime PCR Technologies 3...
Lux Primers
TaqMan Probes, Hydrolysis Probes, 5’-Nuclease Process
Scorpion Primers
HRM
Molecular Beacons
55
55

56. Realtime PCR Instrument 1
Company Instrument Capacity; Labware Type of Thermocycler Robotic Friendly Web Address
(Yes, No, Not
Specified)
StepOne, StepOne+ 48, 96 fast block N
Peltier based, fast
7500 96 well plate N
block option
AB/Life Technologies 96, 384 well plate, or Peltier based, fast appliedbiosystems.com
7900HT Y
TaqMan array block option
96, 384 well plate, or
ViiA 7 fast block Y
TaqMan array
Y; Integration of
24 individually
sample preparation,
BD BD Max controlled chambers; fast cycler bd.com
amplification and
microfluidic cartridge
detection
Sandwich thermal-
Bioer Technology Line-Gene,2,K 33,66,48 tubes N bioer.com.cn/en
electronic Peltier
Biogene Insyte 96 well plate fast cycler NS biogene.com
Bioneer Exicycler 96 well plate Peltier based NS bioneer.com
MiniOpticon, MyIQ2,
Bio-rad 48,96,96,384 well plate Peltier based N bio-rad.com
CFX89, CFX384
16 independent units;
SmartCycler fast cycler N
proprietary tubes
Y; Integration of
1, 4, 16 independent
Cepheid sample preparation, cepheid.com
GeneXpert units; proprietary fast cycler
amplification and
cartridge
detection
48 independent units; Continuous flow fast
GeneXpert Infinity Y 56
proprietary cartridge cycler
56

57. Realtime PCR Instrument 2
Company Instrument Capacity; Labware Type of Robotic Friendly Web Address
Thermocycler (Yes, Not, Not
Specified)
Mastercyler ep
Eppendorf 96 well plate Peltier based N eppendorfna.com
realplex
Evogen Evocycler HD12 12 tubes-proprietary fast cycler N evogen.com
Focus 3M integrated Cycler 96 well disc fast cycler N integratedcycler.com
6 samples-6 tests;
GeneSystems GeneDisc Cycler fast cycler N genesystems.fr
CD format
Peltier based
Illumina Eco Real-Time 48 well plate N illumina.com
36 tubes; 72, 100 fast cycler
Qiagen Rotor-Gene Q N qiagen.com
proprietary tubes
LightCyler V1, V2 32 glass capillaries; N
96, 384 ; 1536 well fast cycler roche-applied-
LightCyler 480; 1536 Y science.com
plate
Roche Diagnostics 2x24 proprietary
Cobas Taqman 48 N
tubes
Peltier based roche.com
4x24 proprietary
Cobas Taqman 96 Y
tubes
Stratagene-Agilent
Mx3000, Mx3005P 96 well plate Peltier based N stratagene.com
Technologies
57
57

58. Innovation in realtime PCR
AlphaHelix QuanTyper
Superconvection and Capilette
Applied Biosystems/Life Technologies
ViiA 7 Realtime PCR with 96, 384 well plate and Taqman Array microfluidic cards
OpenArray with 3072 through hole plate
Fuidigm digital PCR
BioMark Realtime PCR with 48x48 (2304) or 96x96 (9216) Dynamic, Digital and Access
Array IFCs
Roche Applied Sciences
LightCycler 1536 Fast Realtime PCR
Small initiatives like
GeneSystems’s GeneDisc realtime PCR cycler
Enigma Diagnostics
58
58

59. AlphaHelix SuperConvection realtime PCR
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 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.
59
59

60. LifeTechnologies Open Array realtime PCR
OpenArray
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
60
60

61. Fluidigm Digital Array realtime PCR
96.96 Dynamic Array IFC IFC Controller HX BioMArk reader
Digital Array
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.
61
61

62. Roche LC-1536 realtime PCR
1536 ‘regular’ realtime PCR
•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. 62
62

63. 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.
63
63

64. GeneSystems GeneDisc
64
64

65. PCR Instrumentation
So What About Automation?
65
65

66. Popular 96 Well Plate Instrumentation
Difficult to Automate
66
66

67. Fast PCR Popular Instrumentation
67
67

68. Automation with Capillaries?
The “capillary” concern
The “carousel” concern 68
68

69. Fast PCR Instrument: Corbettnow part of Qiagen
Research
Rotor-Gene Q
๏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)
69
69

70. PCR Instrumentation
Solutions for Realtime PCR Automation?
70
70

71. QPCR robotic for 96-384 sample batch
Applied BioSystems PRISM®
7900HT
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
71
71

72. 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 2.5hrs
72
72

73. High Throughput Robotic QPCR with multiple formats
The Roche LighCycler 480
73
73

74. New Step in High Throughput and rapid QPCR
LightCycler 480 and Caliper’s Twister II
The LightCycler 480
74
74

75. Roche LC480 and Automation
75
75

76. Robotic QPCR Process
LC480 and Caliper LS
LC480 and Protedyne Radius
76
76

77. Biomek Nx-Sp8 / LC480 integration
The players: Beckman Coulter, Roche Diagnostics, AIG
An integration from The Biomedical Innovation Platform @
University Hospital of Bordeaux, France with the help of AIG,
Germany.
77
77

78. 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.
78
78

79. PCR Instrumentation
Automation of unconventional realtime PCR
formats?
79
79

80. Automation of PCR Setup with the MagnaPure LCs
MagnaPure LC2 MagnaPure LC
80
80

81. Automation of PCR Setup with the QIAgility
Automated PCR setup in all formats
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
81
81

82. Integration of PCR Setup with the QIAsymphony AS
Automated PCR setup in all formats
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.
82
82

83. Robotic Friendly QPCR Instruments
The Parallab 350
(BioGene) is an
automated nano pipetter,
including a magnetic bead
station/separator and a
fast thermocycler
83
83

84. PCR Instrumentation
Automation Through Integration
84
84

85. 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.
85
85

86. 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.
86
86

87. Cepheid GeneXpert Family-1 to 16 units
87
87

88. A New Step in MDx Automation
Cepheid Infinity / AACC 2008
Up 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.
88
88

89. 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.
89
89

90. Iquum Lab-in-a-Tube Platforms
For example, a 50μl rapid
PCR can be accomplished in
as little as 7 min for 30 cycles.
Liat FlowCycler
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.
90
90

91. 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.
91
91

92. PCR Instrumentation
Automation Through IVD Industry Offering
92
92

93. Realtime PCR: instruments+reagents offers
•Realtime based assay •Abbott Diagnostics •Roche Diagnostics
developments •m2000sp+m2000rt •Cobas Ampliprep +
•Instruments availability •HIV, HBV, HCV, CMV, Taqman 48, 96
EBV, (HSV,VZV), CT/NG •HIV, HBV, HCV
•Industry complete offering •MagnaPureLC2 +LC2?
instrument+reagents •MagnaPure96+LC480?
•Recent increase in rtPCR •CMV, EBV, HSV, VZV
IVD assays •upcoming Cobas 6800
•Cepheid •Gen-Probe/Millipore •Siemens
•GeneExpert, Infinity •Milliprobe •Versant kPCR
•EV, MRSA, GBS •contamination detec. •HIV, HBV, HCV, CT/GC
•Biomerieux •upcoming Panther
•EasyMag+EasyQ •Qiagen
•HIV, Enterovirus, RSV •QIAsymphony SP+AS +
•BD Rotor-Gene Q?
•BD-Max; GBS,NG •upcoming QIAensemble
•Viper SP; CT/GC
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.
93
93

94. Abbott Molecular PCR automation
CMV HBV
HCV Viral Load Viral Load Ct/Ng
Viral Load Screening
HIV Ct
Viral Load Screening
VIROSEQ Open
Génotypage modes
HSV
VZV
Prism 7000 M200RT
94
94

95. Abbott Lab organization solution
Router
LIS
Data
(m2000rt)
Sample Prep
(m2000 sp V3.0) (m2000 rt V2.1)
95
95

96. 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
96
96

97. Roche COBAS TaqMan 96
Robotic
arm
Thermocyclers
Sample racks K-carriers waiting
for TC
97
97

98. Roche Cobas Docking Station
98
98

99. Siemens Molecular - Versant K-PCR
99
99

100. Realtime PCR automation: the new dilemma
Major Diagnostic companies High cost (40-50 €)
reagents Reagents rental programs
Low cost of instruments
Molecular Diagnostic companies Lower cost (20-30 €)
reagents QC, QA, standards
provided
Instruments not provided
Molecular biology companies Unexpensive (<10 €)
reagents QC, QA, standards to
perform
Instruments not provided
100
100

101. Roche Diagnostic Lab. Setup proposition
HIV
HBV
HCV
CE-IVD
HIV 9284
HCV 5552
HBV 1009
CMV 10313
EBV 5104
HSV 2749
VZV 1280
Adenovirus 1 428
HHV6 Quantitatif 1 406
VRS 953
Grippe 894
Enterovirus 725
HHV8 104
BK 100
Parvovirus B19 100
Norovirus 100 CMV
SARS, Coronavirus 50 EBV
Grippe Aviaire H5N1 50
HSV + Home
ParaInfluenzae
Metapneumovirus
608
300 VZV Brew
42109
Home
Brew
101
101

102. Abbott Molecular Lab. Setup proposition
CE-IVD
Evo 75 Nat HSV
VZV
Router
LIS
CE-IVD
HIV 9284
HCV 5552
HBV 1009
CMV 10313 Données
EBV 5104
HSV
VZV
2749
1280
(m2000rt)
HIV
HBV
HCV
CMV
(m2000 rt V2.1) EBV
102
Sample Prep
(m2000 sp V3.0)
35291
102

103. Lab. Setup with M 2000 sp V3.0 et EVO 75
9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 18:00
Lundi Sp1 Extraction HIV 48 tests rt1 PCR HIV 48 tests
Sp2 Extraction HCV 48 tests PCR HCV 48 tests
rt2
Sp1 Extraction CMV 96 tests rt1 PCR CMV 96 tests
Mardi Sp1 Extraction HIV 48 tests rt1 PCR HIV 48 tests
Sp2 Extraction HBV 24 tests rt2 PCR HBV 24 tests
Sp1 extraction EBV 48 tests rt1 PCR EBV 48 tests
Mercredi Sp1 Extraction HIV 48 tests rt1 PCR HIV 48 tests
Sp2 Extraction HCV 48 tests PCR HCV 48 tests
rt2
EVO 75 Ext VZV 24 tests ABI PCR VZV 24 tests
Jeudi Sp1 Extraction HIV 48 tests rt1 PCR HIV 48 tests
Sp1 Extraction CMV 96 tests rt1 PCR CMV 96 tests
EVO 75 Ext HSV 24 tests ABI PCR HSV 24 tests
Sp1 Extraction HIV 48 tests rt1 PCR HIV 48 tests
Vendredi Sp2 Extraction HCV 48 tests
rt2
PCR HCV 48 tests
Sp1 extraction EBV 48 tests rt1 PCR EBV 48 tests
courtesy of A Esnault 2008 Abbott
103
103

104. 104 Beyond HIV,HBV,HCV-CMV,EBV,HSV,VZV
Most comprehensive additional menu in infectious disease:
1) Nanogen-Elitech 2) Qiagen
Adenovirus Flu
HHV6 Enterovirus
Adenovirus 1 428
HHV6 Quantitatif 1 406
VRS Parvovirus B19
VRS 953 Flu SARS, Coronavirus
Grippe 894 Enterovirus H5N1
Enterovirus 725
HHV8 104
HHV8
BK 100 BK
Parvovirus B19 100
Norovirus Cepheid
SARS, Coronavirus 50 AB Prism compatible/CE-IVD AB Prism compatible/CE-IVD
Grippe Aviaire H5N1 50
H5N1 Roche LC compatible
ParaInfluenzae diagenode
Metapneumovirus diagenode
5810
CE-IVD
104

105. Realtime PCR Market Evolution
Major Diagnostic companies - Main molecular markers
(HIV, HBV, HCV, CT/GC)
- CE-IVD, FDA approved
- Instruments
Molecular Diagnostic reagents -Numerous minor markers
companies -Rarely major markers
-ASR, RUO
-Rarely instruments
Recently -Full menu
major diagnostic companies -CE-IVD
enhanced menu for minor -Instruments
makers
105
105

106. 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..)
✓ Realtime PCR arrays and gene expression
106
106

107. 107
107