2011 course on Molecular Diagnostic Automation - Part 2 - Amplification


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2011 course on Molecular Diagnostic Automation - Part 2 - Amplification.
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2011 course on Molecular Diagnostic Automation - Part 2 - Amplification

  1. 1. Patrick Merel Biomedical Innovation Platform (PTIB), Pessac, France pmerel@mac.comPCR Automation in Molecular Diagnostic part 2 1 1
  2. 2. From Waterbath to PCR ChipsDNA diagnostic mostly PCR diagnosticPCR challengers: small market share but robust alternativesAutomation 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. 3. Siemens Branched DNA (bDNA) bDNA Invader NASBA/TMA 3 3
  4. 4. Siemens bDNA latest Automated System Bayer Versant 440 4 4
  5. 5. Siemens bDNA Assay Workflow 5 5
  6. 6. bDNA in-house Full Automation 6 6
  7. 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 thecomplexity of these other test methods.•Minimal required specimen preparation, allowing samples to be processed quicklyand efficiently.•Protection against cross contamination problems that can occur with some targetamplification assays.•Hybrid Capture tests are available to detect human papillomavirus (HPV), chlamydiatrachomatis (CT), neisseria gonorrhea (GC), and cytomegalovirus (CMV); the latter isavailable in tube format only. 7 7
  8. 8. Digene’s Hybrid Capture Technology 8 8
  9. 9. Digene’s Hybrid Capture Technology 9 9
  10. 10. Hybrid Capture Next Generation Platforms 10 10
  11. 11. Signal Amplification with the Invader reaction IF the variation or sequence in question is present, anIn 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 patentedassociate with a specific region of DNA to Cleavase® enzymes specifically cleave the primary probesgenerate 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 absenceinterrogated. of the specific target, no flap is released. 11 11
  12. 12. Signal Amplification with the Invader reactionReleased 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 eserve as Invader® oligos in a second, reactions occur simultaneously,simultaneous invasive cleavage reaction on they can produce 1 million to 10a labeled, synthetic oligo, the fluorescence million labeled cleavage productsresonance energy transfer (FRET) probe. per target sequence. A standard 4-Cleavage of this FRET probe results in the hour reaction produces over 10generation of a fluorescent signal. million-fold signal amplification. 12 12
  13. 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. 14. Invader Signal Amplification EvolutionInvader 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® chemistrybrings 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. 15. Target Amplification by TMATranscription Mediated Amplification: TMA 15 15
  16. 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. 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. 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. 19. Automating PCR OperationsPlasticware options Tubes and plates format Sealing platesInstrumentation for regular PCR robotic friendly? integration friendly?Instrumentation for realtime PCR robotic friendly? integration friendly?PCR offering in the IVD market 19 19
  20. 20. Microtiterplate formats for PCR Various format of 96 well plate format Up to 384 and 1536 well plate format 20 20
  21. 21. Fluidigm, Illumina, LifeTech: New Formats? Fuidigm EP1 or BioMArk 96.96 Dynamic Array IFC 48.770 Digital Array IFC Digital ArrayProducing up to 9,216 real-time qPCR reactions inthe same amount of time as a single 384-well plate,while using just 1/200th the amount of reagents. 21 21
  22. 22. Fluidigm, Illumina, LifeTech: New Formats? 48-well polypropylene reaction plates and Optically clear pressure- sensitive adhesive film Back to the 48’sThe Eco thermal system incorporates a precisely electroformed hollow silver block that is heated and cooled by asingle Peltier device. The hermetically sealed hollow block contains a conductive fluid and two opposing agitatorsdriven by electromagnetic motors. During PCR cycling, these agitators rapidly circulate the fluid throughout thehollow block, transferring heat from the Peltier device evenly across the block. This unique design virtuallyeliminates 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. 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. 24. Innovation in ‘regular’ PCRAlphaHelix 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. 25. Innovation in ‘regular’ PCR Icubate: iCubate Cassettes are custom made toautomate molecular protocols such asPCR, ligation, hybridization and detection.Possible applications include pathogenidentification from various sample sources. An iCubate system includes one Reader,one Processor (4 units), and a computerwith preinstalled software. The internal andexternal barcode scanners locatedthroughout the system will ensure error freeprocessing and tracking of your samples. Proprietary ARM-PCR technology(Amplicon Rescued Multiplex PCR) allowsmultiple targets to be amplified in onereaction. Proven array detection technologyanalyzes multiple targets at once. Plug and play operation, no need for Icubate:extensive training. Up to twelve units can be linked togetherto run up to 48 samples simultaneously. 25 25
  26. 26. Innovation in ‘regular’ PCR Idaho Technologies: FilmArray: The FilmArray integratessample preparation, amplification,detection, and analysis into one easy touse system capable of massivelymultiplexed PCR. Fully automated,multiplex PCR capable of detectinggreater than 100 targets in one sample inless than one hour. Inject unprocessed sample and water, start the run Closed system prevents cross-contaminationand walk away Multiplexed testing analyzes up to 120 tests per System integrates sample preparation, amplification, sampledetection and analysis Rapid results in 1 hour from sample injection All necessary reagents are freeze dried in one pouchand are stable at room temperature 26 26
  27. 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. 28. High Throughput PCR wanted ๏Increasing number of tests ๏Increasing number of samples 28 28
  29. 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. 30. Thermocyclers for a complete robotic integrationü Automation of lid movementsü Automation of PCR programsü Biorad Tetradü AB 9700 AutoLidü MWG Primus HTü Biometra Trobotü ThermoHybaid 30 30
  31. 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. 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. 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 MJRWhat 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. 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. 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*03/07Four 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. 36. Going further with Thermocycler Integration 36 36
  37. 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. 38. Thermocycler Modules and Robotic Integration Integration of a remote PCR block on a MultiProbe robotic workstation from Perkin Elmer 38 38
  39. 39. Thermocyclers and High-End Robotic Integration epMotion Eppendorf Workstation 39 39
  40. 40. Thermocyclers and High-End Robotic Integration PSS, Niagala Bio-Station FDX 40 40
  41. 41. Thermocyclers and High-End Robotic IntegrationRoche Diagnostic Cobas Taqman96 integration through the DockingStation to the Cobas Amplirep. 41 41
  42. 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. 43. Full Robotic Integration๏Automation of plate transportation๏Automation of lid movements๏Automation of PCR programs๏Biorad Tetrad 43 43
  44. 44. Full Robotic IntegrationAutomation of plate transportationAutomation of lid movementsAutomation of PCR programsThermoHybaid, Bio-Rad 44 44
  45. 45. Full Robotic IntegrationAutomation of plate transportationAutomation of lid movementsAutomation of PCR setupAutomation of PCR programsVarious robotic arms available 45 45
  46. 46. Accessories for Full AutomationPlate sealersRobotic arms or grippers 46 46
  47. 47. Accessories for Full Automation Plate sealer Plate piercerVelocity11s PlateLoc Thermal Plate Sealer.As a complementary machine to the PlateLoc, thePlatePierce pierces a wide variety of seals on 96or 384 well microplates in only 4 seconds of cycletime. 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. 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. 49. LabAutomation 2002 Show Room 49 49
  50. 50. Protedyne BioCube Workstation The Protedyne PCR BioCube™ isthe 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 outputconveyors for unattended operation. 50 50
  51. 51. Protedyne BioCube WorkstationProtedyne has also very recently The Protedyne PCR BioCube announced the integration of with AB-9700 thermocyclersRoche LC480 thermocycler in the BioCube. 51 51
  52. 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. 53. Fast PCR with integrated detection: the revolutionThe Lightcycler integrates a 24 sample micro-volume fluorimeter with an air thermal cyclerdelivering accurate and high-speed temperaturecontrol.The Lightcycler is the fastest thermal cycler in the world. Thirty cycle reactions canbe run in as little as six minutes while an integrated fluorimeter monitors reactionsas they occur. The Lightcycler eliminates the need to perform both amplification andseparate product analysis allowing unprecedented throughput. 53 53
  54. 54. RealTime PCR Technologies 1 & 2 54 54
  55. 55. RealTime PCR Technologies 3... Lux PrimersTaqMan Probes, Hydrolysis Probes, 5’-Nuclease Process Scorpion Primers HRM Molecular Beacons 55 55
  56. 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 optionAB/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. 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 plateRoche Diagnostics 2x24 proprietary Cobas Taqman 48 N tubes Peltier based roche.com 4x24 proprietary Cobas Taqman 96 Y tubesStratagene-Agilent Mx3000, Mx3005P 96 well plate Peltier based N stratagene.com Technologies 57 57
  58. 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 PCRSmall initiatives like GeneSystems’s GeneDisc realtime PCR cycler Enigma Diagnostics 58 58
  59. 59. AlphaHelix SuperConvection realtime PCRAlphaHelix QuanTyper 48: the first qPCR instrument to utilize SuperConvection, making it the worlds 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. 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. 61. Fluidigm Digital Array realtime PCR 96.96 Dynamic Array IFC IFC Controller HX BioMArk readerDigital ArrayProducing up to 9,216 real-time qPCR reactions inthe same amount of time as a single 384-well plate,while using just 1/200th the amount of reagents. 61 61
  62. 62. Roche LC-1536 realtime PCR1536 ‘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. ofjust 0.5-2.0 µl. 62 62
  63. 63. GeneSystems GeneDisc CyclerThe GeneDisc uses a hybrid technology between the DNA chip and real-timePCR microplates that proves to be especially relevant for routine diagnosticapplications.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 reagentsnecessary 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 thebacterial DNA samples to the reaction chambers. 63 63
  64. 64. GeneSystems GeneDisc 64 64
  65. 65. PCR InstrumentationSo What About Automation? 65 65
  66. 66. Popular 96 Well Plate InstrumentationDifficult to Automate 66 66
  67. 67. Fast PCR Popular Instrumentation 67 67
  68. 68. Automation with Capillaries?The “capillary” concern The “carousel” concern 68 68
  69. 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. 70. PCR InstrumentationSolutions for Realtime PCR Automation? 70 70
  71. 71. QPCR robotic for 96-384 sample batchApplied 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. 72. QPCR Robotic IntegrationHigh Throughput Core Systemsalready exist for PCRautomationCombination of BCI coresystem + Prism 7900 allowsconfigurations for:NA extractionPCR setupPlate sealingPrism 7900 loadingUp to 384 PCR every 2.5hrs 72 72
  73. 73. High Throughput Robotic QPCR with multiple formats The Roche LighCycler 480 73 73
  74. 74. New Step in High Throughput and rapid QPCR LightCycler 480 and Caliper’s Twister II The LightCycler 480 74 74
  75. 75. Roche LC480 and Automation 75 75
  76. 76. Robotic QPCR ProcessLC480 and Caliper LS LC480 and Protedyne Radius 76 76
  77. 77. Biomek Nx-Sp8 / LC480 integrationThe players: Beckman Coulter, Roche Diagnostics, AIGAn integration from The Biomedical Innovation Platform @University Hospital of Bordeaux, France with the help of AIG,Germany. 77 77
  78. 78. Very High Throughput QPCR with the LightCycler 1536•Increase throughput fourfold.•Generate 1,536 data points in a singlerun in less than 50 minutes.•Use significantly less reagent andsample with reaction volumes ofjust 0.5-2.0 µl. 78 78
  79. 79. PCR InstrumentationAutomation of unconventional realtime PCR formats? 79 79
  80. 80. Automation of PCR Setup with the MagnaPure LCsMagnaPure LC2 MagnaPure LC 80 80
  81. 81. Automation of PCR Setup with the QIAgilityAutomated 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. 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. 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. 84. PCR InstrumentationAutomation Through Integration 84 84
  85. 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. 86. Cepheid GeneXpertThe GeneXpert System is a 1 to 4-site, random accessinstrument integrating real-time amplification and detectionfeatures seen in the SmartCycler System, but deliveringresults from unprocessed samples in less than 30 minutes.The internal module is the common technology link betweenthe SmartCycler and GeneXpert, performing real-timeamplification and detection.The GeneXpert automates sample preparation, integratingthe complex steps of DNA extraction in the microfluidiccartridges. 86 86
  87. 87. Cepheid GeneXpert Family-1 to 16 units 87 87
  88. 88. A New Step in MDx Automation Cepheid Infinity / AACC 2008Up to 2,074 test results in a mere 24 hour period. Less than 52minutes time to first result. Less than 2 minutes sample prep timeper test. MRSA, EV, GBS tests available. 88 88
  89. 89. Iquum Lab-in-a-Tube PlatformLiat 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. 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. 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. Its PCR Real Time format will accept FDA cleared, analyte specific reagents (ASR) and/or your homebrew assays. HandyLabs proprietary unitized reagents and microfluidics cartridge radically reduce the chance of a contamination event in your laboratory. 91 91
  92. 92. PCR InstrumentationAutomation Through IVD Industry Offering 92 92
  93. 93. Realtime PCR: instruments+reagents offers•Realtime based assay •Abbott Diagnostics •Roche Diagnosticsdevelopments •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, VZVIVD 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. 94. Abbott Molecular PCR automation CMV HBV HCV Viral Load Viral Load Ct/Ng Viral Load Screening HIV CtViral Load ScreeningVIROSEQ OpenGénotypage modes HSV VZV Prism 7000 M200RT 94 94
  95. 95. Abbott Lab organization solution Router LIS Data (m2000rt) Sample Prep(m2000 sp V3.0) (m2000 rt V2.1) 95 95
  96. 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. 97. Roche COBAS TaqMan 96 Robotic armThermocyclers Sample racks K-carriers waiting for TC 97 97
  98. 98. Roche Cobas Docking Station 98 98
  99. 99. Siemens Molecular - Versant K-PCR 99 99
  100. 100. Realtime PCR automation: the new dilemmaMajor Diagnostic companies High cost (40-50 €) reagents Reagents rental programs Low cost of instrumentsMolecular 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. 101. Roche Diagnostic Lab. Setup proposition HIV HBV HCV CE-IVDHIV 9284HCV 5552HBV 1009CMV 10313EBV 5104HSV 2749VZV 1280Adenovirus 1 428HHV6 Quantitatif 1 406VRS 953Grippe 894Enterovirus 725HHV8 104BK 100Parvovirus B19 100Norovirus 100 CMVSARS, Coronavirus 50 EBVGrippe Aviaire H5N1 50 HSV + HomeParaInfluenzaeMetapneumovirus 608 300 VZV Brew 42109 Home Brew 101 101
  102. 102. Abbott Molecular Lab. Setup proposition CE-IVD Evo 75 Nat HSV VZV Router LIS CE-IVDHIV 9284HCV 5552HBV 1009CMV 10313 DonnéesEBV 5104HSVVZV 2749 1280 (m2000rt) HIV HBV HCV CMV (m2000 rt V2.1) EBV 102 Sample Prep (m2000 sp V3.0) 35291 102
  103. 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 18:00Lundi 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 testsMardi 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 testsMercredi 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 testsJeudi 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 testsVendredi 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. 104 Beyond HIV,HBV,HCV-CMV,EBV,HSV,VZV Most comprehensive additional menu in infectious disease: 1) Nanogen-Elitech 2) Qiagen Adenovirus Flu HHV6 EnterovirusAdenovirus 1 428HHV6 Quantitatif 1 406 VRS Parvovirus B19VRS 953 Flu SARS, CoronavirusGrippe 894 Enterovirus H5N1Enterovirus 725HHV8 104 HHV8BK 100 BKParvovirus B19 100Norovirus CepheidSARS, Coronavirus 50 AB Prism compatible/CE-IVD AB Prism compatible/CE-IVDGrippe Aviaire H5N1 50 H5N1 Roche LC compatibleParaInfluenzae diagenodeMetapneumovirus diagenode 5810 CE-IVD 104
  105. 105. Realtime PCR Market EvolutionMajor 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. 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 singleinstrument 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
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