Chipron Array R.A. Hamidjaja (Cib/LZO)
Advantage of array? <ul><li>More specific than electrophoresis (sequence based selection) </li></ul><ul><li>Ability to pro...
Array format <ul><li>Planar array </li></ul><ul><li>Suspension array </li></ul>Luminex Chipron
Workflow Suspension array Planar array
Improvement  (compared to Luminex) <ul><li>Faster PCR step: from 4 hours to 1 hour 15 min </li></ul><ul><li>2 markers for ...
Evaluation & Validation <ul><li>Specificity: 120 DNA samples (incl. eukaryotes) </li></ul><ul><li>Approximate LOD (limit o...
Specificity B. anthracis Brucella Y. pestis C. burnetii F. tularensis B
Specificity Ixodes Anas platyrhyncos (Wild duck) Lepus europaeus  (Brown hare) Y. pseudotuberculosis Homo sapiens E. coli ...
LOD (triplicate) Rough estimation of genomic DNA mass from 1 bacteria: 5 fg 0 0 3 3 3 2YPcaf 0 1 3 3 3 3YPpla-02 0 0 0 3 3...
Dynamic range Ft: F. tularensis Yp: Y. pestis
Lesson learned (1) <ul><li>Idea: Speed up PCR reaction using superconvection </li></ul>AmpXpress thermocycler AlphaHelix
SuperConvection <ul><li>Convection: major mode of heat & mass transfer that occurs due temperature differences which creat...
Possible   Explanations <ul><li>Martensson & Skote et al. 2006 describes the theoretical en numerical analysis of SuperCon...
Rayleigh number and convection <ul><li>Convection can be predicted using Rayleigh number (Ra) </li></ul><ul><li>Ra =  ρ 0 ...
Epilogue <ul><li>Personal observation & experience: </li></ul><ul><li>Increasing reaction vessel’s surface to volume ratio...
Lesson learned (2) <ul><li>False positive results? </li></ul><ul><li>Possibilities: </li></ul><ul><li>Hybridization of uns...
Things to do <ul><li>Consensus on data analysis </li></ul><ul><li>Manuscript for publication </li></ul><ul><li>Step over t...
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20110201 Chipron2011 Linked In

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Chipron Array

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20110201 Chipron2011 Linked In

  1. 1. Chipron Array R.A. Hamidjaja (Cib/LZO)
  2. 2. Advantage of array? <ul><li>More specific than electrophoresis (sequence based selection) </li></ul><ul><li>Ability to produce more information from 1 sample (Not restricted on the quantity of available detection channel) </li></ul>Disadvantage of array? <ul><li>Qualitative results (end-point signal detection) </li></ul><ul><li>Post PCR method (Never as fast as qPCR) </li></ul>
  3. 3. Array format <ul><li>Planar array </li></ul><ul><li>Suspension array </li></ul>Luminex Chipron
  4. 4. Workflow Suspension array Planar array
  5. 5. Improvement (compared to Luminex) <ul><li>Faster PCR step: from 4 hours to 1 hour 15 min </li></ul><ul><li>2 markers for Brucella added: from 16-plex to 18-plex </li></ul><ul><li>User friendly method </li></ul><ul><li>Cheap hardware: ‘Lidl’ quality slides scanner needed compared to a 2 laser flow cytometer </li></ul><ul><li>Chromometric signal detection: signals intensity not significantly affected by time </li></ul>
  6. 6. Evaluation & Validation <ul><li>Specificity: 120 DNA samples (incl. eukaryotes) </li></ul><ul><li>Approximate LOD (limit of detection): serial dilutions of genomic DNA, 10000 fg-1fg </li></ul><ul><li>Dynamic range: Genomic DNA from 2 organisms with different concentrations </li></ul>
  7. 7. Specificity B. anthracis Brucella Y. pestis C. burnetii F. tularensis B
  8. 8. Specificity Ixodes Anas platyrhyncos (Wild duck) Lepus europaeus (Brown hare) Y. pseudotuberculosis Homo sapiens E. coli B. cereus F. tularensis A B. thuringiensis
  9. 9. LOD (triplicate) Rough estimation of genomic DNA mass from 1 bacteria: 5 fg 0 0 3 3 3 2YPcaf 0 1 3 3 3 3YPpla-02 0 0 0 3 3 1YPyin Harbin 0 0 1 3 3 3YPypo Y. pestis 0 0 0 0 0 2FTpdn 0 0 0 0 0 1FTpdf-B 0 0 3 3 3 1FTwbk 3 3 3 3 3 2FTisf BD07-537 0 0 3 3 3 3FTfoa F. tularensis 0 0 0 2 3 1CBicd 0 2 3 3 3 1CBis1 0 0 3 3 3 1CBser 9 mile 0 0 3 3 3 1CBcom C. burnetii 0 0 0 0 0 1Brwbo-02 0 0 0 0 0 1Brwbo 0 1 3 3 3 1Brbcs-02 ATCC 23457 0 1 3 3 3 1Brbcs Br. melitensis 3 3 3 3 3 2BAcab 0 0 3 3 3 2BAcya-02 Vollum 493 1 0 3 3 3 1BApl3 B. anthracis 1 fg 10 fg 100 fg 1000 fg 10000 fg Probes Organism
  10. 10. Dynamic range Ft: F. tularensis Yp: Y. pestis
  11. 11. Lesson learned (1) <ul><li>Idea: Speed up PCR reaction using superconvection </li></ul>AmpXpress thermocycler AlphaHelix
  12. 12. SuperConvection <ul><li>Convection: major mode of heat & mass transfer that occurs due temperature differences which create density stratification in the liquid </li></ul><ul><li>In theory increase of gravity through centrifugation: </li></ul><ul><li>Improve heat transfer thus shorten ramp time </li></ul><ul><li>Enhance mass/particles movement thus (Coriolis force) improve reaction kinetics </li></ul><ul><li> Overal effect: Faster PCR </li></ul>In our case: To get the same LOD and dynamic range, faster PCR is not possible Conventional thermocycler 1 X g Natural convection AmpXpress thermocycler 7000 X g Superconvection
  13. 13. Possible Explanations <ul><li>Martensson & Skote et al. 2006 describes the theoretical en numerical analysis of SuperConvection phenomenon using the following experimental set up: </li></ul><ul><ul><li>100 ul reaction volume (ours: 25 ul) </li></ul></ul><ul><ul><li>Thermocycling conditions: 95 o C-70 o C (ours: 95 o C-57 o C) </li></ul></ul><ul><ul><li>Singleplex PCR (ours: 18-plex PCR) </li></ul></ul><ul><ul><li>Assumption: reaction viscosity & thermal transfer characteristic = water characteristic </li></ul></ul><ul><ul><li>Assumption: Coreolis force play dominant role during centrifugation as to enhance particle movement into a stable movement pattern </li></ul></ul><ul><li>PCR reaction have higher viscosity and different thermal transfer characteristic than water (eg: DMSO in the PCR kit, high concentration of DNA etc.) </li></ul><ul><li>Lower reaction volume means less density stratification in the liquid </li></ul><ul><li>Ma & Xu et al. 1998 conclude that Coreolis force only plays dominant role under specific conditions </li></ul><ul><li> SuperConvection may not occur in our assay  Overal effect: slow PCR </li></ul>Do not forget!!! Enzyme activity determines the final reaction speed
  14. 14. Rayleigh number and convection <ul><li>Convection can be predicted using Rayleigh number (Ra) </li></ul><ul><li>Ra = ρ 0 g α ∆ TL 3 </li></ul><ul><li> ______________ </li></ul><ul><li> κμ </li></ul><ul><li>ρ 0 = average liquid density </li></ul><ul><li>g = gravity </li></ul><ul><li>α = coefficient of thermal expansion </li></ul><ul><li>∆ T = temperature difference across liquid </li></ul><ul><li>L = liquid depth </li></ul><ul><li>κ = thermal diffusivity </li></ul><ul><li>μ = dynamic viscosity </li></ul> Ra = 0; no convection  Ra >>; the higher the rate of convection Significant contributor !!!
  15. 15. Epilogue <ul><li>Personal observation & experience: </li></ul><ul><li>Increasing reaction vessel’s surface to volume ratio (miniaturization): </li></ul><ul><li>Decrease thermal capacity </li></ul><ul><li>Increase heat transfer rate </li></ul><ul><li> Effect: Faster PCR </li></ul>
  16. 16. Lesson learned (2) <ul><li>False positive results? </li></ul><ul><li>Possibilities: </li></ul><ul><li>Hybridization of unspecific PCR prod. </li></ul><ul><li>Hybridization of primer dimers </li></ul>Cause: Biotin contamination of the capture probes Solution: If possible purchase the capture probes from other manufacturer Negative control PCR prod Water
  17. 17. Things to do <ul><li>Consensus on data analysis </li></ul><ul><li>Manuscript for publication </li></ul><ul><li>Step over to NGS?? </li></ul>

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