Für Thomas Greinke

Detection of genetic modifications
in raw and processed cotton products
• Lothar Kruse PhD
• Workshop „Cotton“, Berlin, February 2009
Impetus GmbH & Co. Bioscience KG
•
•
•
•
•
Accredited according to DIN EN ISO/IEC 17025: 2000
15 years of experience with DNA technology
Currently 8 employees
Focus: Detection of animal species and genetic
modifications in food, feed, seed, animal and plant fibers
Located in Bremerhaven
Possible applications
Meat, raw
Meat, cooked
Sausage
Canned meat
Feedstuff
Bonemeal
Bloodmeal
Animalmeal
Petfood
Stock cube
Lard
Gelatin
Fish, raw
Fish, smoked
Canned fish
Seeds
Soyabeans/-meal/-whole-meal
Corn/-meal/- whole-meal
Pollen
GMO-detection
Animal and plant-species-identification
Milk
full-cream
Milk
UHT
Cheese
Plant and animal
fibres in raw
material and fabrics
Furs
Chocolate
Cornflakes
Honey
Juice
Marzipan
Tofu
Soya-drink
Soya-bread
Soya-goulash
Soya-sauce
Soya-lecithine
Soya-oil, raw
Margarine
Corn-starch
Ketchup
Cottonseed-oil,
raw
From biological samples to PCR-products
Schematic view of cells
Animal cell
Plant cell
Eucaryotic DNA
PCR-cycle
Start: Template-DNA
72°C
Elongation
3
Reaction runs
in thermocycler
1
Denaturation
95°C
50-60°C
Annealing
2
Anzahl PCR-Zyklen
Effectiveness of PCR
Exponential increase of copy number
Anzahl doppelsträngiger DNA-Moleküle
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
0
0
2
4
8
16
32
64
128
256
512
1.024
2.048
4.096
8.192
16.384
32.768
65.536
131.072
262.144
524.288
1.048.576
2.097.152
4.194.304
8.388.608
16.777.216
33.554.432
67.108.864
134.217.728
268.435.456
536.870.912
1.073.741.824
2.147.483.648
4.294.967.296
8.589.934.592
17.179.869.184
34.359.738.368
68.719.476.736
137.438.953.472
274.877.906.944
549.755.813.888
1.099.511.627.776
2.199.023.255.552
4.398.046.511.104
8.796.093.022.208
17.592.186.044.416
35.184.372.088.832
70.368.744.177.664
140.737.488.355.328
281.474.976.710.656
Screening- / Construct- / Eventspecific detection
Screening
CaMV 35S-promoter
Plant-DNA
CaMV 35S-promoter
Nos-terminator
CTP2
Cry-gene
Bt cottoncotton-35S/CTP2
Construct-specific detection
Nos-terminator
Plant-DNA
NosNos-plant
Event-specific detection
PCR-Precautions
•
•
•
•
Strictly separated working areas for DNA-extraction, PCR set-up and detection
Appropriate air-ventilation / Air-condition/ Air filter-system
Solutions in aliquots, One-way gloves, Filter-tips, overall etc.
Intensive and frequent cleaning
•
•
•
•
•
Analyses are performed as double-reactions
Positive-controls
Negative-controls
Inhibition-controls
Plus/Minus-results are re-analysed
Facts about gm cotton
• Global cultivation area: 35 million hectares
• Genetically modified: 15 mio. ha, i. e. 43%
• India: 9,4 mio. ha (6.2 mio. ha gm, 76%); China: 5,6 mio. ha (3.8 mio.
ha gm, 66%); USA: 4.4 mio. ha (4.0 mio. ha gm, 86%); Argentina: 0.4
mio. ha (0.38 mio. ha gm, 95%); Mexico: 0.12 mio. ha (0.065 mio. ha
gm, 57%)
• More than 20 gm cotton events worldwide
• Herbicide tolerance (RR cotton) and/or insect resistance (Bt cotton)
• 6 events have been approved for food and feed in the EU: MON1445,
MON531, MON15985, MON531xMON 1445,
MON15985xMON1445, LLCotton 25
Why should be tested?
• The rapidly expanding cultivation of gm cotton will make it harder to
get „conventional“ cotton
• The use of genetically modified plants for feed, food and cultivation
is strictly regulated (labelling, thresholds) in the EU (1829/2003,
1830/2003)
• Gm cotton fibres used in textiles are not affected by this regulations –
but labels like organic, bio, kbA etc. exclude the use of gm cotton
(zero tolerance)
• Contamination and adulteration is already common – in more than
30% of all cotton samples labelled as „non gm“ we have analysed so
far genetic modifications could be identified
Specific PCR identification
untreated
washed
mechan. treated
chem. treated
bleached
stained
Cotton
+
+
+
+
+
+
Hemp
+
+
+
Flax
+
+
+/-
+
+
Nettle
+
+
+/-
-
-
Ramie
+
+
-
+/-
+/-
Kenaf
+
-
-
-
-
Sheep (Wool)
+
+
+
+
Cashmere goat
+
+
+
+
Yak
+
+
+/-
+/-
Camel
+
+
+
+/-
+, detectable; -, not detectable; +/-, not always detectable; grey : unusual processing steps
TaqMan-Principle
1. Sequence-specific annealing
of probe and PCR-primers
2. Primer-extension and probe-hydrolysis
3. Release of reporter dye
4. Reporter-signal inreases proportionally
to number of cycles
Influence of heat treatment on the DNA-fragment-length
Lane 1: DNA-Standard (λ-DNA, Hind III-Digest)
Lane 2: Bovine-DNA, untreated, 2 µg
Lane 3: Bovine-DNA, 5 min 100°C, 2 µg
Lane 4: Bovine-DNA, 15 min 100°C, 2 µg
Lane 5: Bovine-DNA, 30 min 100°C, 2 µg
Lane 6: Bovine-DNA, 5 min 121°C, 2 µg
Lane 7: Bovine-DNA, 13 min 121°C, 2 µg
Lane 8: DNA-Standard (pUC18, Hpa II-Digest)
TaqMan-Principle
1. Sequence-specific annealing
of probe and PCR-primers
2. Primer-extension and probe-hydrolysis
3. Release of reporter dye
4. Reporter-signal inreases proportionally
to number of cycles