Multiplex arrays for Genotyping Multiplex arrays for Genotyping 2 - - PowerPoint PPT Presentation

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Multiplex arrays for Genotyping Multiplex arrays for Genotyping 2 - - PowerPoint PPT Presentation

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Multiplex arrays for Genotyping Multiplex arrays for Genotyping 2 Alere Technologies, Jena 3 4 Complex information Complex information 5 Jena & Zeiss 6 Arrays and reader systems Clondiag: Clondiag: 7 S. Aureus product development:

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SLIDE 1

Multiplex arrays for Genotyping Multiplex arrays for Genotyping

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SLIDE 2

Alere Technologies, Jena

2

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SLIDE 3

3

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SLIDE 4

Complex information Complex information

4

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SLIDE 5

Jena & Zeiss

5

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SLIDE 6

Clondiag: Clondiag: Arrays and reader systems

6

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SLIDE 7
  • S. Aureus product development:

7

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SLIDE 8

VLA Products

  • Detects ~57 antimicrobial resistance genes in Gram
  • Detects ~57 antimicrobial resistance genes in Gram

negative bacteria

  • Detects ~92 virulence genes in E. coli
  • Detects ~95 virulence factors and toxin genes
  • Detects ~100 virulence factors and antimicrobial

resistance genes in Gram positive bacteria resistance genes in Gram positive bacteria

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SLIDE 9

P bli i Publications

AMR A T b

  • AMR-ve ArrayTube

Development of a miniaturised micro-array for the rapid identification of antimicrobial resistance genes in Gram-negative g g bacteria

Miranda Batchelor, Katie L Hopkins, Ernesto Liebana, Peter Slickers, Ralf Ehricht Muriel Mafura Frank Aarestrup Dik Mevius Felicity Clifton-Hadley Ehricht, Muriel Mafura, Frank Aarestrup, Dik Mevius, Felicity Clifton Hadley, Martin J Woodward, Rob H Davies, E John Threfall, Muna F Anjum; International Journal of Antimicrobial Agents 2008 May 31(5) p 440-451

  • Ec ArrayTube

P th t i E h i hi li b i i i t i d DNA i Pathotyping Escherichia coli by using miniaturised DNA microarrays

Muna F Anjum, Muriel Mafura, Peter Slickers, Karin Ballmer, Peter Kuhnert, Martin J Woodward, Ralf Ehricht; Applied and Environmental Microbiology, pp gy 2007 Sept 73(17), p.5692-7

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SLIDE 10

Packs contain: Packs contain:

  • 25 ArrayTubes
  • 25 ArrayTubes
  • 5 foil packets containing 5 tubes in each

E h t b h i b (AT L t)

  • Each tube has unique number (AT Lot)

Primer mix

  • Primer mix
  • Freeze-dried

R tit t d i 120 l t

  • Reconstituted in 120µl water

IFU d li t

  • IFU and gene list
  • Labelling and hybridisation protocol
  • Buffers and reagent ordering information
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SLIDE 11

Buffers and reagents

  • Not supplied

Ordering information provided in IFU

  • Ordering information provided in IFU
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SLIDE 12

Buffers and reagents

R i tli d i IFU

  • Recipes outlined in IFU
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SLIDE 13

D t A l i

B2304(01-2571) B2305(NCTC50270) B2306(NCTC50073) B2307(NCTC50273) B2308(E111592) B2309(00-419) B2310(NCTC50022) B2311(P429859) B2312(P2530920) Probe Rep 1 Rep 2 PCR Rep 1 Rep 2 PCR Rep 1 Rep 2 PCR Rep 1 Rep 2 PCR Rep 1 Rep 2 PCR Rep 1 Rep 2 PCR Rep 1 Rep 2 P CR Rep 1 Rep 2 P CR Rep 1 Rep 2 P CR prob_aac3Ia_1 0.00 0.00 0.03 0.00 0.01
  • 0.01
  • 0.01
  • 0.02
  • 0.01
  • 0.10
0.03 0.00
  • 0.02
0.00
  • 0.03
0.03 0.00
  • 0.03

Data Analysis

p _ _ prob_aac3IVa_1 0.04 0.20
  • 0.02
0.00 0.00
  • 0.01
  • 0.11
  • 0.03
  • 0.01
  • 0.10
0.01 0.01
  • 0.03
  • 0.02
  • 0.03
0.06 pos 0.00
  • 0.06
prob_aac6Ib_1
  • 0.01
0.02 0.00 0.00 0.00 0.00 0.00
  • 0.01
1.00 1.06 pos 0.03 0.00
  • 0.02
  • 0.03
  • 0.05
  • 0.01
0.00 0.04 prob_aadA1_1 0.89 0.57 pos 0.00 0.01 0.00
  • 0.01
  • 0.05
  • 0.02
0.99 0.99 pos 0.01 0.00
  • 0.02
0.02
  • 0.04
  • 0.01
0.58 0.30 pos prob_aadA2_1 0.50 0.07 pos 0.23 0.04 0.00
  • 0.01
  • 0.01
0.02 0.71 0.25 pos 0.28 0.00 pos
  • 0.01
  • 0.02
  • 0.04
  • 0.02
0.02
  • 0.02
prob_aadA4_1
  • 0.01
  • 0.01
0.00
  • 0.01
0.00
  • 0.01
0.01
  • 0.02
0.02
  • 0.09
0.98 1.27 pos
  • 0.01
0.00
  • 0.05
0.00 0.00 0.06 prob_acc1_11
  • 0.02
0.01 0.02 0.00 0.00
  • 0.01
0.07 0.00 0.00
  • 0.07
0.02 0.00 0.00 0.04
  • 0.04
0.02 0.00 0.06 prob_acc2_11
  • 0.02
0.00 0.02 0.00 0.01
  • 0.01
0.02
  • 0.01
  • 0.01
  • 0.09
0.01 0.00
  • 0.02
  • 0.02
  • 0.05
  • 0.02
0.00 0.08 prob_act1_11
  • 0.02
0.00 0.04 0.00 0.02 0.00 0.07 0.03 0.00
  • 0.07
0.03 0.00
  • 0.01
0.00
  • 0.03
0.03 0.00
  • 0.01
prob_ant2Ia_1 0.02 0.01 0.00 0.00 0.00 0.00 0.03
  • 0.01
0.17 0.04
  • 0.01
0.00
  • 0.02
0.01
  • 0.04
0.02 0.00 0.01 prob_catA1_11 0.01 0.01
  • 0.01
0.00 0.00
  • 0.01
0.00
  • 0.03
1.00 1.05 pos 0.01 0.00
  • 0.02
0.00 1.02 1.02 pos 1.08 6.45 pos prob_catB3_11
  • 0.01
0.00 0.02 0.00 0.00 0.00
  • 0.03
  • 0.02
0.00
  • 0.06
0.00 0.00
  • 0.02
  • 0.01
  • 0.04
0.00 0.00
  • 0.05
prob_catB8_12 0.00 0.01 0.02 0.00 0.02
  • 0.01
0.00 0.02 0.00
  • 0.12
0.01 0.00 0.00
  • 0.02
  • 0.04
0.00 0.01
  • 0.12
prob_catIII_1 0.02
  • 0.01
  • 0.02
0.01 0.00 0.01 0.06
  • 0.01
  • 0.01
  • 0.07
  • 0.01
0.00 0.85 0.25
  • 0.04
0.01 0.00
  • 0.10
prob_cmlA1_11 0.97 1.15 pos 0.02 0.00 0.01
  • 0.01
  • 0.13
  • 0.03
0.00
  • 0.08
  • 0.01
0.00
  • 0.02
  • 0.01
  • 0.03
0.02 0.00 0.03 prob_cmy_11 0.10 0.01 0.20 0.00 0.15
  • 0.01
  • 0.11
  • 0.01
0.16
  • 0.05
0.16 0.00
  • 0.03
0.01 0.03 0.04 0.05 0.00 prob_ctxM1_11 0.00
  • 0.01
0.00 0.00 0.01
  • 0.01
0.02
  • 0.02
  • 0.01
  • 0.12
  • 0.01
  • 0.01
  • 0.02
  • 0.02
  • 0.06
0.01 0.02
  • 0.03
prob_ctxM1_12
  • 0.01
0.01 0.01 0.00 0.00 0.01
  • 0.10
  • 0.03
0.00
  • 0.13
  • 0.01
0.00
  • 0.02
0.00
  • 0.04
  • 0.04
0.00 0.02 prob_ctxM2_11 0.02 0.00 0.02 0.00
  • 0.01
0.00 0.00 0.01
  • 0.01
  • 0.06
0.01 0.00
  • 0.02
0.00
  • 0.04
0.09 0.00 0.13 prob_ctxM9_11 0.00 0.00 0.00 0.00 0.00
  • 0.02
  • 0.14
  • 0.02
0.00
  • 0.14
0.01 0.00
  • 0.02
  • 0.04
  • 0.04
0.01 0.00
  • 0.04
prob_dfr12_11 0.83 0.09 pos 0.02 0.00 0.01
  • 0.01
0.03
  • 0.01
0.76 0.09 pos 0.02 0.00
  • 0.03
  • 0.01
  • 0.02
0.07 0.00
  • 0.01
prob_dfr13_11
  • 0.01
  • 0.01
0.00 0.00 0.00
  • 0.01
0.05
  • 0.01
0.00
  • 0.08
0.01 0.00
  • 0.03
0.00
  • 0.05
  • 0.01
0.00 0.10 prob_dfrA1_21
  • 0.02
  • 0.01
0.03 0.00 0.01
  • 0.01
  • 0.02
0.00 0.99 0.86 pos 0.00 0.00
  • 0.01
  • 0.01
  • 0.05
0.02 0.00 0.02 prob_dfrA1_22 0.02
  • 0.01
0.00 0.00 0.01
  • 0.01
  • 0.02
  • 0.01
0.29
  • 0.05 pos
0.00 0.00
  • 0.03
0.01
  • 0.05
0.02 0.00 0.04 prob_dfrA14_21 0.02
  • 0.01
0.04 0.00 0.03
  • 0.01
  • 0.08
  • 0.01
0.02
  • 0.08
0.01 0.00
  • 0.02
0.01
  • 0.03
0.01 0.35
  • 0.05 pos
prob_dfrA15_1
  • 0.02
  • 0.01
0.01 0.00 0.01 0.00 0.03
  • 0.02
0.01
  • 0.10
  • 0.01
0.00
  • 0.03
0.00
  • 0.05
  • 0.02
0.00 0.00 prob_dfrA17_11 0.03 0.00 0.07 0.00 0.03
  • 0.01
0.04
  • 0.03
0.03
  • 0.14
1.01 1.24 pos
  • 0.03
  • 0.03
0.42 0.50 pos 0.01 0.04 prob_dfrA19_1
  • 0.02
0.01 0.00 0.00 0.00
  • 0.02
  • 0.10
  • 0.02
  • 0.01
  • 0.17
0.42 0.12
  • 0.02
0.01
  • 0.06
  • 0.02
0.00 0.02 prob_dfrA7_11 0.01
  • 0.01
0.02 0.00 0.00
  • 0.01
  • 0.04
  • 0.02
0.00
  • 0.09
0.00 0.00
  • 0.03
0.00 0.93 0.96 pos 0.00 0.05 prob_dfrA7_12 0.00
  • 0.03
0.00 0.00 0.00
  • 0.01
0.24
  • 0.02
0.00
  • 0.08
0.20 0.03
  • 0.02
0.00 0.95 0.97 pos 0.00
  • 0.01
prob_dfrV_21 0.03
  • 0.01
0.06 0.01 0.01
  • 0.01
  • 0.03
0.01 0.01
  • 0.08
0.05 0.01
  • 0.01
  • 0.04
  • 0.04
0.05 0.00 0.04 prob_dha1_1 0.02
  • 0.03
0.02 0.00 0.01
  • 0.01
0.05
  • 0.01
0.00
  • 0.07
  • 0.02
0.00
  • 0.03
0.00
  • 0.03
  • 0.02
0.00 0.00 prob_floR_11
  • 0.01
0.00 0.95 0.97 pos 0.01 0.02 0.05 0.00 0.00
  • 0.08
0.98 1.02 pos
  • 0.02
0.02
  • 0.04
0.00 0.00
  • 0.05
prob_fox_11 0.03 0.00 0.01 0.00 0.01 0.01 0.00
  • 0.01
0.00
  • 0.08
  • 0.01
0.00
  • 0.02
0.01
  • 0.04
0.00
  • 0.01
0.04 prob_intI2_11 0.01
  • 0.03
  • 0.01
0.00 0.00
  • 0.01
  • 0.01
  • 0.03
0.92 0.83 pos 0.00 0.00
  • 0.02
  • 0.01
  • 0.04
  • 0.02
0.00 0.00 prob_len1_11 0.04 0.00 0.07 0.02 0.04 0.02 0.01 0.05 0.00 0.18 0.05 0.01 0.08 0.05 0.02 0.09 0.00 0.11 prob_mox_1mm
  • 0.01
  • 0.02
0.03 0.00 0.00
  • 0.01
0.04
  • 0.03
  • 0.01
  • 0.06
0.01
  • 0.01
  • 0.03
0.02
  • 0.07
0.00 0.00 0.04 prob_mox_1pm 0.01
  • 0.01
0.02 0.00 0.00
  • 0.01
  • 0.13
  • 0.02
0.00
  • 0.08
0.01 0.00
  • 0.03
  • 0.03
  • 0.05
  • 0.04
0.00 0.03 prob_oxa1_21
  • 0.03
  • 0.04
0.03 0.00 0.02 0.00
  • 0.05
0.03
  • 0.01
  • 0.12
0.00
  • 0.01
  • 0.01
0.03
  • 0.04
0.06
  • 0.02
  • 0.11
prob_oxa2_11 0.01 0.00 0.01 0.01 0.01
  • 0.01
  • 0.05
0.01
  • 0.01
  • 0.12
0.00 0.00
  • 0.03
0.00
  • 0.04
0.01 0.00
  • 0.04
prob_oxa7_11 0.02
  • 0.01
0.04 0.00 0.00
  • 0.02
0.00
  • 0.03
  • 0.01
  • 0.20
  • 0.01
0.00
  • 0.02
  • 0.01
  • 0.06
0.01
  • 0.01
0.08 b 9 11 0 02 0 05 0 00 0 00 0 00 0 01 0 03 0 01 1 01 0 96 0 02 0 00 0 02 0 02 0 04 0 01 0 00 0 04 prob_oxa9_11 0.02
  • 0.05
0.00 0.00 0.00
  • 0.01
  • 0.03
  • 0.01
1.01 0.96 pos 0.02 0.00
  • 0.02
  • 0.02
  • 0.04
0.01 0.00 0.04 prob_per2_1 0.00 0.02
  • 0.02
0.00 0.00
  • 0.01
  • 0.06
  • 0.02
0.03
  • 0.09
  • 0.01
0.00
  • 0.02
  • 0.01
  • 0.04
0.14 0.00
  • 0.11
prob_qnr_11
  • 0.02
0.00 0.08
  • 0.01
0.00
  • 0.01
0.01
  • 0.02
  • 0.01
  • 0.07
0.01 0.00
  • 0.02
  • 0.02
  • 0.05
  • 0.01
  • 0.01
  • 0.08
prob_qnr_12 0.00
  • 0.01
0.04 0.00 0.02 0.00 0.03 0.07 0.01
  • 0.04
  • 0.01
  • 0.01
0.05 0.02
  • 0.03
0.01 0.00 0.06 prob_qnrB_11 0.03 0.02 0.01
  • 0.01
0.00
  • 0.01
  • 0.03
  • 0.01
0.00
  • 0.05
0.04
  • 0.01
  • 0.03
  • 0.04
  • 0.03
0.03 0.00 0.03 prob_qnrB_12 0.03
  • 0.01
0.08 0.01 0.03 0.01 0.10 0.02 0.00
  • 0.04
0.01 0.00 0.03 0.00
  • 0.01
0.02
  • 0.01
  • 0.01
prob_qnrS_11
  • 0.01
0.02
  • 0.02
0.00 0.00
  • 0.01
  • 0.04
0.00 0.00
  • 0.04
  • 0.01
  • 0.01
  • 0.02
  • 0.01
  • 0.03
  • 0.03
0.00
  • 0.06
prob_shv1_11 0.04 0.01 0.02 0.00 0.01
  • 0.01
0.00 0.00 0.89 0.29 pos
  • 0.01
0.01
  • 0.01
  • 0.05
  • 0.02
0.01 0.00 0.00 prob_strA_611 0.26 0.03 pos
  • 0.03
  • 0.01
0.16 0.08 0.00
  • 0.02
  • 0.01
  • 0.07
  • 0.01
  • 0.01
0.10 0.00 0.03 0.10 0.02 0.04 prob_strB_611 0.98 1.18 pos 0.20
  • 0.01
0.95 1.01 pos
  • 0.12
  • 0.01
  • 0.01
  • 0.09
  • 0.02
  • 0.01
1.00 2.39 pos 0.94 1.02 pos 0.57 3.30 pos prob sul1 11 0 03 0 00 0 95 0 98 0 00 0 01 0 08 0 03 0 99 1 01 pos 0 97 1 17 pos 0 04 0 01 0 82 0 90 pos 0 86 2 75 pos prob_sul1_11
  • 0.03
0.00 0.95 0.98 0.00
  • 0.01
0.08
  • 0.03
0.99 1.01 pos 0.97 1.17 pos
  • 0.04
0.01 0.82 0.90 pos 0.86 2.75 pos prob_sul2_11
  • 0.01
  • 0.02
0.00 0.00 0.00
  • 0.01
  • 0.03
  • 0.04
0.00
  • 0.12
  • 0.01
0.00
  • 0.03
0.02
  • 0.01
0.04 pos 0.59 0.20 pos prob_sul3_11 1.03 1.19 pos 0.00 0.00 0.00
  • 0.01
0.02
  • 0.03
0.00
  • 0.10
0.01 0.00
  • 0.03
0.01
  • 0.05
0.00
  • 0.01
  • 0.10
prob_tem1_1 1.02 1.23 pos 0.25 0.01 0.22 0.01 0.04 0.04 1.01 1.08 pos 1.01 1.29 pos 0.01 0.03 1.04 1.05 pos 1.15 11.50 pos prob_tetA_11 0.00
  • 0.01
0.00 0.00 0.00
  • 0.01
  • 0.03
0.00
  • 0.01
  • 0.08
0.00 0.00
  • 0.03
  • 0.02
0.72 0.71 pos 0.01
  • 0.06
prob_tetB_11 0.95 0.96 pos 0.00
  • 0.01
0.97 0.92 pos 0.02
  • 0.03
0.98 0.88 pos
  • 0.01
0.00
  • 0.03
  • 0.01
  • 0.04
0.00 0.90 0.95 pos prob_tetC_11
  • 0.01
  • 0.01
  • 0.01
0.00 0.00
  • 0.01
0.01
  • 0.04
  • 0.01
  • 0.11
0.00 0.01
  • 0.03
0.01
  • 0.05
0.00 0.00 0.00 prob_tetD_1
  • 0.01
  • 0.01
  • 0.01
0.00 0.01
  • 0.01
0.01 0.02 0.00
  • 0.06
  • 0.02
0.00
  • 0.02
0.01
  • 0.04
0.00
  • 0.01
  • 0.03
slide-14
SLIDE 14

Analysis y

slide-15
SLIDE 15

2008: 2008: Feedback from potential users

  • To create a product range with a broader portfolio

Eas to se standardi ed b ffer s stem

  • Easy to use standardized buffer system
  • Automated result report
  •  cooperation between Alere and VLA started in

 cooperation between Alere and VLA started in 2009

slide-16
SLIDE 16
  • S. Aureus Genotyping
  • S. Aureus Genotyping

Analysis of 333 Markers

positive Metal mark positive negative negative

slide-17
SLIDE 17

The Arraymate reader

C b d f

  • Can be used for

Strips and Tubes

  • Automated

Software Analysis

slide-18
SLIDE 18

Automated Analysis of the Array

slide-19
SLIDE 19

Test report Test report

slide-20
SLIDE 20
  • S. Aureus Genotyping report

D \ d \PC RDR\D kt \Id ti

  • D:\andreas.voss\PC_RDR\Desktop\Identi

bac\Genelists\S. aureus report 2 lt ht 2.result.res.htm

  • ..\Genelists\S. aureus

report.result B.res.htm p _

slide-21
SLIDE 21

Identibac Kits

An Overview of the Test Protocol

slide-22
SLIDE 22

id tib www.identibac.com

22

slide-23
SLIDE 23

23

slide-24
SLIDE 24

24

slide-25
SLIDE 25

e.g.: S. aureus Genotyping Kit

slide-26
SLIDE 26

Publications Publications

  • Rapid genotyping of methicillin-resistant Staphylococcus aureus (MRSA)

isolates using miniaturised oligonucleotide arrays

  • S. Monecke and R. Ehricht; Clinical Microbiology and Infectious Diseases

2005 Oct 11(10) p825 833 2005 Oct, 11(10), p825 – 833

  • Microarray based study on virulence-associated genes and resistance

determinants of Staphylococcus aureus isolates from cattle Stefan Monecke, Peter Kuhnert, Helmut Hotzel, Peter Slickers and Ralf Ehricht; Veterinary Microbiology 2007 Nov 15 125)1-2), p128 – 140. Epub 2007 May y gy ) ), p p y 24

  • Assignment of Staphylococcus aureus isolates to clonal complexes based

Assignment of Staphylococcus aureus isolates to clonal complexes based

  • n microarray analysis and pattern recognition

Stefan Monecke, Peter Slickers and Ralf Ehricht; FEMS Immunology Medical Microbiology 2008 Jul 53(2), p237-51. Epub 2008 May 27

slide-27
SLIDE 27

Principle of the Principle of the

  • S. aureus Genotyping Kit

333 diff t DNA S th A St i 333 different DNA Sequences on the ArrayStrip

many markers => 1 test

27

slide-28
SLIDE 28

The Workflow The Workflow

28

slide-29
SLIDE 29

Cl i d H t f B t i Cloning and Harvest of Bacteria

Loop empty Loop full

29

slide-30
SLIDE 30

Cell Lysis and RNA Degradation Cell Lysis and RNA Degradation

buffer A1 protein pellet A2 p p

  • cell wall lysis
  • RNAse
  • add 0.2 mL buffer A1 to protein pellet A2
  • add an inoculating loop of bacteria

30

g

slide-31
SLIDE 31

DNA Extraction – Qiagen DNeasy

  • incubate 30 min at 37 °C/ 550 rpm
  • proceed with first step of Qiagen DNeasy kit

31

slide-32
SLIDE 32

DNA Extraction – Qiagen DNeasy

1-2 hours

  • follow the instructions

from Qiagen Kit

20 min 32

slide-33
SLIDE 33

Trouble shooting!

  • most problems trace back to poor DNA quality

NOT

  • DNA is NOT exponetial PCR amplified

33

slide-34
SLIDE 34

Recommended: Measure DNA

  • A260 method
  • Agarose gel

34

slide-35
SLIDE 35

L b l DNA Label DNA

slide-36
SLIDE 36

l“ li PCR „real“ vs. linear PCR

linear PCR

i nucleotides

l S DNA

linear PCR (StaphyType)

primer 5‘ 3‘ DNA 5‘ 3‘

  • only S.a. DNA

is copied

„real“ PCR

primer nucleotides 5‘ 3‘ DNA

  • also copies

primer nucleotides 5‘ 3‘

p are copied

slide-37
SLIDE 37

ft 2 PCR l ... after 2 PCR cycles ...

linear PCR

  • only S.a. DNA

linear PCR (StaphyType)

5‘ 3‘ DNA 5‘ 3‘

  • nly S.a. DNA

is copied 1*2 = 2 copies 1 2 2 copies

  • also copies

are copied

„real“ PCR

5‘ 3‘ DNA

are copied 22 = 4 copies

5‘ 3‘

2 = number of PCR cycles

slide-38
SLIDE 38

real“ vs linear PCR

PCR l li PCR PCR 600000

„real vs. linear PCR

PCR cycle linear PCR PCR 1 1 1 2 2 2 3 3 4 4 4 8 500000

„real“ PCR

4 4 8 5 5 16 6 6 32 7 7 64 300000 400000 plification 8 8 128 9 9 256 10 10 512 11 11 1024 200000 300000

  • fold amp

12 12 2048 13 13 4096 14 14 8192 15 15 16384 100000

linear PCR

16 16 32768 17 17 65536 18 18 131072 19 19 262144 5 10 15 20 PCR cycle

(StaphyType)

20 20 524288

slide-39
SLIDE 39

„real“ vs. linear PCR

„real“ PCR amplifies the DNA much more !!!!! => for the S Aureus genotyping test: => for the S. Aureus genotyping test:

  • Pros: no significant danger of cross contamination
  • Cons: no signal from poor DNA !!!!!!!
slide-40
SLIDE 40

Biotin labelling with linear PCR

i nucleotides Biotin label

2 hours

primer 5‘ 3‘ staphylococcal DNA

333 different primers for different sequences alongside the genome for different sequences alongside the genome

slide-41
SLIDE 41

Components for Biotin labelling Components for Biotin labelling

333 primers Biotin label enzyme (Taq Polymerase) customer‘ s DNA Biotin label etc. (Taq Polymerase) DNA

slide-42
SLIDE 42

L b lli i Th l Labelling in Thermocycler

slide-43
SLIDE 43

bind labelled DNA bind labelled DNA to array

slide-44
SLIDE 44

the array - undeveloped

DNA spot Metal mark p

slide-45
SLIDE 45

activation of the arrays

  • wash with water

wash with water

  • wash with C1
slide-46
SLIDE 46

Binding to Arrays = Hybridisation

  • add C1 to the labelling product
  • add mixture to activated arrays

y

  • shake 1 hour at 55°C
slide-47
SLIDE 47

binding = hybridisation

1 hour

array surface capture DNA molecule

slide-48
SLIDE 48

wash away unbound fragments

array surface capture DNA molecule

5 i 5 min

slide-49
SLIDE 49

Binding of Binding of Horseradish Peroxidase

10 min

Streptavidin‐HRP

array surface

Biotin within the DNA binds to St t idi th t i li k d t H di h P id Streptavidin that is linked to Horseradish Peroxidase

slide-50
SLIDE 50

Horseradish Peroxidase ...

  • Combine C3 and C4
slide-51
SLIDE 51

Horseradish Peroxidase ...

  • add to the arrays
  • 10 min 30 °C

10 min 30 C

  • NEVER too hot
  • NEVER too hot

(55°C) !!!!!

slide-52
SLIDE 52

Wash away unbond material

array surface

5 i 5 min

slide-53
SLIDE 53

t i d d t stain and read out

slide-54
SLIDE 54

D1 = substrate of D1 substrate of Horseradish Peroxidase

slide-55
SLIDE 55

Horseradish Peroxidase Horseradish Peroxidase  converts D1

5 min

array surface

slide-56
SLIDE 56

the array – developed

positive Metal mark positive negative negative

slide-57
SLIDE 57

Reading with the Array reader

slide-58
SLIDE 58

Latest EHEC Outbreak in Germany

The enemy in food EHEC EHEC: the birth of a new id i epidemie

slide-59
SLIDE 59

Wh M lti l G t i ? Why Multiplex Genotyping? Identify down to strain level

  • EHEC Outbreak in Europe Mai/June 2011:
  • Rare Strain of E. Coli Unknown in Previous Outbreaks

Rare Strain of E. Coli Unknown in Previous Outbreaks

  • ScienceDaily (June 2, 2011) —

The strain of enterohaemorrhagic Escherichia coli (EHEC) 0104:H4 isolated from cases in the EHEC infection

  • utbreak in Germany is a rare one, seen in humans before,

but never in an EHEC outbreak. This has been confirmed by the World Health Organization (WHO) Collaborating Centre for Reference and Research on Escherichia and Centre for Reference and Research on Escherichia and Klebsiella, the Statens Serum Institut in Denmark.

slide-60
SLIDE 60

Influenza Outbreaks

  • Avian flu, Swine flu, seasonal flu or mixed

Avian flu, Swine flu, seasonal flu or mixed infection?

  • 2011:
  • 2011:
  • More swine flu deaths last winter than during

pandemic pandemic

  • More people died after contracting swine flu in

Britain last winter than during the previous Britain last winter than during the previous year’s pandemic

  • The Telegraph 13th of June
slide-61
SLIDE 61

MRSA Superbug? Resistance and virulence factors

MRSA can be multiresistant and

  • MRSA can be multiresistant and

multivirulent

  • Recombination and mutation can

change the risks

  • Need to identify the strain details
slide-62
SLIDE 62

Arrays

Id tib S A G t i id tifi

  • Identibac S. Aureus Genotyping identifies

333 different genetic markers!

  • Identibac Influenza Genotyping identifies

all 16 H- & 9 N types in a single test! all 16 H- & 9 N types in a single test!

  • Identibac E coli Genotyping can
  • Identibac E. coli Genotyping can

detect 92 virulence genes in Escherichia coli

  • Identibac AMR-ve Genotyping characterise

54 antimicrobial resistance genes in gram negatives 54 antimicrobial resistance genes in gram negatives.

slide-63
SLIDE 63

For more information:

id tib

  • www.identibac.com
  • www.alere-technologies.com
  • Info@identibac com

Info@identibac.com

  • Andreas.Voss@Alere.com
slide-64
SLIDE 64

Thank you for your attention!

A Q ti ?

  • Any Questions?
  • More Information via e-mail?
  • Interested in updates of new Products?
  • Your suggestion for new Genes on existing Array?

Your suggestion for new Genes on existing Array?

  • Customised arrays for your Institut?

Ad d P d t T i i i J ?

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