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??