20110201 Chipron2011 Linked In
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Transcript of 20110201 Chipron2011 Linked In
1 01 February 2011
Chipron Array
R.A. Hamidjaja (Cib/LZO)
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Advantage of array?1. More specific than electrophoresis (sequence based selection)
3. Ability to produce more information from 1 sample (Not restricted on the quantity of available detection channel)
Disadvantage of array?1. Qualitative results (end-point signal detection)
3. Post PCR method (Never as fast as qPCR)
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Array format
● Planar array
● Suspension array
Luminex
Chipron
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Workflow
Suspension array
Planar array
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Improvement (compared to Luminex)
● Faster PCR step: from 4 hours to 1 hour 15 min
● 2 markers for Brucella added: from 16-plex to 18-plex
● User friendly method
● Cheap hardware: ‘Lidl’ quality slides scanner needed compared to a 2 laser flow cytometer
● Chromometric signal detection: signals intensity not significantly affected by time
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Evaluation & Validation
1. Specificity: 120 DNA samples (incl. eukaryotes)
3. Approximate LOD (limit of detection): serial dilutions of genomic DNA, 10000 fg-1fg
5. Dynamic range: Genomic DNA from 2 organisms with different concentrations
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Specificity
B. anthracis Brucella
Y. pestisC. burnetiiF. tularensis B
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Specificity
Ixodes
Anas platyrhyncos
(Wild duck)
Lepus europaeus (Brown hare)
Y. pseudotuberculosis
Homo sapiens
E. coliB. cereus F. tularensis A
B. thuringiensis
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LOD (triplicate)
003332YPcaf
013333YPpla-02
000331YPyinHarbin
001333YPypoY. pestis
000002FTpdn
000001FTpdf-B
003331FTwbk
333332FTisfBD07-537
003333FTfoaF. tularensis
000231CBicd
023331CBis1
003331CBser9 mile
003331CBcomC. burnetii
000001Brwbo-02
000001Brwbo
013331Brbcs-02ATCC 23457
013331BrbcsBr. melitensis
333332BAcab
003332BAcya-02Vollum 493
103331BApl3B. anthracis
1 fg10 fg100 fg1000 fg10000 fgProbesOrganism
Rough estimation of genomic DNA mass from 1 bacteria: 5 fg
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Dynamic range
Ft: F. tularensis
Yp: Y. pestis
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Lesson learned (1)
● Idea: Speed up PCR reaction using superconvection
AmpXpress thermocycler
AlphaHelix
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SuperConvection● Convection: major mode of heat & mass transfer that occurs due
temperature differences which create density stratification in the liquid
Conventional thermocycler
1 X g
Natural convection
In theory increase of gravity through centrifugation:
• Improve heat transfer thus shorten ramp time
• Enhance mass/particles movement thus (Coriolis force) improve reaction kinetics
Overal effect: Faster PCR
In our case:
To get the same LOD and dynamic range, faster PCR is not possible
AmpXpress thermocycler
7000 X g
Superconvection
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Possible Explanations● Martensson & Skote et al. 2006 describes the theoretical en numerical analysis of
SuperConvection phenomenon using the following experimental set up:– 100 ul reaction volume (ours: 25 ul)– Thermocycling conditions: 95 oC-70 oC (ours: 95 oC-57 oC)– Singleplex PCR (ours: 18-plex PCR)– Assumption: reaction viscosity & thermal transfer characteristic = water
characteristic– Assumption: Coreolis force play dominant role during centrifugation as to
enhance particle movement into a stable movement pattern
1. PCR reaction have higher viscosity and different thermal transfer characteristic than water (eg: DMSO in the PCR kit, high concentration of DNA etc.)
2. Lower reaction volume means less density stratification in the liquid
3. Ma & Xu et al. 1998 conclude that Coreolis force only plays dominant role under specific conditions
SuperConvection may not occur in our assay Overal effect: slow PCR
Do not forget!!!
Enzyme activity determines the
final reaction speed
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Rayleigh number and convection● Convection can be predicted using Rayleigh number (Ra)
Ra = ρ0gα∆TL3 ______________ κμ
ρ0 = average liquid density
g = gravityα = coefficient of thermal expansion∆T = temperature difference across liquidL = liquid depthκ = thermal diffusivity
μ = dynamic viscosity
Ra = 0; no convection
Ra >>; the higher the rate of convection
Significant contributor !!!
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Epilogue
Personal observation & experience:
Increasing reaction vessel’s surface to volume ratio (miniaturization):
4. Decrease thermal capacity
5. Increase heat transfer rate
Effect: Faster PCR
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Lesson learned (2)● False positive results?
Negative control
PCR prod
Possibilities:
2. Hybridization of unspecific PCR prod.
3. Hybridization of primer dimers
Water
Cause:
Biotin contamination of the capture
probes
Solution:
If possible purchase the capture probes from other manufacturer
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Things to do
● Consensus on data analysis
● Manuscript for publication
● Step over to NGS??