LAMP-BART detection of GM

markers in-planta

?

Presented by Guy KiddleL u m o r a

•Gene of Interest

• Promoter (e.g.. NOS)

• Terminator (e.g. 35S)

• Selectable marker (e.g. GFP)

Plant Transformation Technology

Ecological demands

(Increased Biomass)

Agricultural

Sustainability

(Resistance)

“Pro et Contra”

Food quality

(Phyto-ceuticals)

Research

(Gene function)

FRANKINSTIEN FOOD ??

lobby / concern

INTENSIVE?INTENSIVE?ORGANIC?ORGANIC?

CHOICE / REGULATION

Lumora's

Technology

DNA replication

BART Reaction Scheme

PPi + APS ATP + SO4-

ATP sulphurylase

AMP + PPiATP + Luciferin O2 CO2+ ++Luciferase

LAMP-BART

Initiate

Propogate

intermediate

PPi

Simple execution

Dispense

Heat

Detect

Dedicated BART Instruments

Static (CCD Camera / heater)

Portable (Photodiodes / heater)

BART Output

Evolution of light from

incubated samples

Tmax

Light detection

ABI Prism 7000

R.A.P.I.D. IDAHO

GM LAMP-BART

Specificity &

Sensitivity

0

5

10

15

20

25

30

1 6 11 16 21 26 31 36

20

25

30

35

40

2000 (0.%5)

2000 (1%)

5000 (2%)

4000 (5%)

35S-p

ADH ref

qPCR Analysis of Bt11 maize GM reference series

0

5

10

15

1 6 11 16 21 26 31 36

0

5

10

15

20

25

30

1 6 11 16 21 26 31 36

300 (0.1%)

2000 (0.%5)

300 (0.1%)

2000 (0.%5)

2000 (1%)

5000 (2%)

4000 (5%)

NOS-t

Rn

Cycle

0

5

10

15

20

25

30

35

0 0.1 0.5 1 2 5

Ct

QPCR Ct values for GM titration series

ADH CAMV 35S-p NOS-t

0, 0.1, 0.5, 1, 2, 5 %, 0,0.1,0.5,1 2, 5 % 0,0.1,0.5,1 2, 5 %

0 0.1 0.5 1 2 5

ADH 35s NOSt

1200

1400

1600

1800

0

200

400

600

800

1000

1200

1400

1600

1 11 21 31 41 51 61 71 81

All 105

104 (5%)

5 x 103 (2%)

2 x 103 (1%)

900 (0.5%)

ADH1

35s

LAMP-BART Analysis of Bt11 maize GM reference seriesIntensity

0

200

400

600

800

1000

1 11 21 31 41 51 61 71 81

900 (0.5%)

200 (0.1%)

NOSt

0

200

400

600

800

1000

1200

1400

1 11 21 31 41 51 61 71 81

104 (5%)

5 x 103 (2%)

2 x 103 (1%)

900 (0.5%)

200 (0.1%)

Intensity

0

10

20

30

40

50

60

70

0 0.1 0.5 1 2 5

ADH 35s NOSt

Tm

ax

LAMP-BART Tmax values for GM titration series

ADH 35s NOSt

ADH CAMV 35S-p NOS-t

0, 0.1, 0.5, 1, 2, 5 %, 0,0.1,0.5,1 2, 5 % 0,0.1,0.5,1 2, 5 %

Transformation Events

Zea mays

Variety of trans-elements

Brassica napus

0

200

400

600

800

1000

1200

1400

1600

1800

1 11 21 31 41 51 61 71 81

Bt11 (5%)

ADH

35S

NOSt

1200

1400

1600

1800Roundup (5%)

ADH

35S

NOSt

Specificity of GM LAMP-BARTintensity

0

200

400

600

800

1000

1 11 21 31 41 51 61 71 81

0

200

400

600

800

1000

1200

1400

1600

1800

1 11 21 31 41 51 61 71 81

Mono810 (100%)

ADH35S

NOSt

ADH

intensity

DNA Quality

affects affects

LAMP-BART

λ DNA standard 5 to 50ng

Promega Wizard

Nucleon Phytopure

DNA integrity and quantification

NanoDrop

CTAB

0, 0.1, 0.5, 1 2, 5 %

Comparison of genomic DNA

quantifications (density vs. spectra)

200

250

0

50

100

150

Promega Phytopure CTAB

Gel NanoDrop

ng/ ul

DNA Quality vs. Amplification Linearity & Reproducibility

0

10

20

30

40

50

60

70

80

90

Phytopure0

10

20

30

40

50

60

70

80

90

Promega Wizard 35S-p NOSt

70

80

90

CTAB

LAMP-BART QT-PCR

copy No

1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06

0

10

20

30

40

50

60

70

1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+01 1.E+02

10x10x

Point of Use

Simplified genome prep

Sample boiled in saline + CHELEX

Sample desalted

React BART Analysis

ADH1

35s

All 105

2 x 103 (2%)

5 x 103 (5%)

1 x 103(1%)

5 x 102(0.5%)

Portable solution for GM detection (PDQ)

NOSt

5 x 102(0.5%)

102(0.1%)

2 x 103 (2%)

5 x 103 (5%)

1 x 103(1%)

5 x 102(0.5%)

102(0.1%)

90

100

Linearity of CHELEX-Saline extracted DNA in LAMP (10min DNA prep)

qPCR

0

10

20

30

40

50

60

70

80

10 100 1000 10000

CHELEX - SALINE

35S

NOSt

Summary

GM LAMP-BART

A simple rapid technique

Quantifiable

Portable

Sensitive & reproducible

Specific

Good value

Acknowledgements

L u m o r a

Jim Murray,

Manuela Rizzoli,

Cathy Moore

Jim Murray,

Manuela Rizzoli,

Cathy Moore