item 7. sampling and lmos detection 7.3. identification of lmos
DESCRIPTION
Item 7. SAMPLING AND LMOs DETECTION 7.3. IDENTIFICATION OF LMOs. Detection is required when :. By law in the country is required identification and/or labelling Mixtures between GMOs + non-GMOs Need to export to a country with strict legislation Need to verify non-GMOs shipments - PowerPoint PPT PresentationTRANSCRIPT
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Item 7. SAMPLING AND LMOs DETECTION
7.3. IDENTIFICATION OF LMOs
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Detection is required when:
• By law in the country is required identification and/or labelling
• Mixtures between GMOs + non-GMOs• Need to export to a country with strict
legislation• Need to verify non-GMOs shipments• For environmental risk verifications
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In the international market• Important focus
differences between commercial blocks:– USA does not require
identification and GM crops are easily set free into the environment
– EU requires labelling with 0.9% treshold
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• First GM-food authorized 1994First GM-food authorized 1994• They were labelled… and people bought them!!!They were labelled… and people bought them!!!• The variety lost sensory characteristics and was retired from The variety lost sensory characteristics and was retired from the marketthe market
“ “Historical” cans of GM tomato puree with long shelf lifeHistorical” cans of GM tomato puree with long shelf life
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Sampling
Extraction of:
Protein
DNA
PCR end point
RTq-PCR
Immunostrips
ELISA plates
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In order to detect proteins, specific antibodies are required. The antibodies are proteins with quaternary structure.
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Control line. Antibodies anti-IgG adsorbed
Test line. Antibodies anti-antigen adsorbedAntibodies anti-antigen
conjugated with enzyme
Lateral flow test, IMMUNOSTRIP
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The antibodies are binded to their antigen in the sample and the complex antigen-antibody moves by
capillarity towards the reaction lines.
Lateral Flow test,IMMUNOSTRIP
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Antigen binds to the antibodies that are in the test line.
Free antibodies bind to the antibodies anti-Ig present in the control line.
Lateral Flow test,IMMUNOSTRIP
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Immunostrip procedure
1. Weigh 250 mg of fresh leaf (plantlet)
2. Insert the sample in the bag
3. Grind or crush the sample
4. Insert the immunostrip into the bag with SEB or MEB buffer.
5. Let stand for 10 minutes (vertical position)
6. Read results
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Qualitative immunoassay in strip.
Ej. Cry9C QuickStixTM Envirologix
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IMMUNOSTRIP Cry 1Ab/Ac
Maíz BT 11
10% 1%0.5%
0.1%
Also by this technique can be detected CP4-EPSPS protein in samples with low % of transgenic maize
IMMUNOSTRIP CP4 EPSPS
Maíz NK603
10% 1%0.1% 0.05
%
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The first Ac is adsorbed to the plate
ELISA:
Enzyme-Linked-ImmunoSorbent Assay
Format: DAS (Double Antibody Sandwich)
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Sample that contains protein is added
Protein
ELISA:
Enzyme-Linked-ImmunoSorbent Assay
Formato: DAS (Double Antibody Sandwich)
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Second antibody is added. It is conjugated with a enzyme
Enzyme
Antibody
ELISA:
Enzyme-Linked-ImmunoSorbent Assay
Format: DAS (Double Antibody Sandwich)
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Colorless
compound
Colorless
compoundProduct Product
Enzyme
Antibody
ELISA:
Enzyme-Linked-ImmunoSorbent Assay
Formato: DAS (Double Antibody Sandwich)
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ELISA plate, second antibody congujated to alkaline phospatase
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Quantitative Immunoassay (ELISA)
Sample extraction.Centrifugation to clarify samples
Preparation of ELISA plates
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Sample applicationIncubation at room temperature
Wash
Substrate addition/ Colour development Spectrophotometric
measurement - for quantification-
ELISA cont.
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Specificity assesment of antibodies in the ELISA test
1 2 3
A
B
C
D
E
F
G
H
1 2 3 A
B
C
D
E
F
G
H
1 2 3
A MEB Cry 1Ab/1Ac NK 603
B MEB Cry 1Ab/1Ac NK 603
C CP4 Cry 3A Bt 11
D CP4 Cry 3A Bt 11
E NPT II Cry 3Bb1 MON 810
F NPT II Cry 3Bb1 MON 810
G Cry 2A Cry 1C Chalqueño
H Cry 2A Cry 1C Chalqueño
KIT CP4 EPSPSKIT Cry 1Ab/1Ac
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Quantitative Analysis by ELISA in detection of protein CP4-EPSPS
1 2 3 4 5
A MEB 0.125 % Mon810 10% Nk603 10% Bt11 10%
B MEB 0.125 % Mon810 10% Nk603 10% Bt11 10%
C 1% 0.062 % Mon810 1% Nk603 1% Bt11 1%
D 1% 0.062 % Mon810 1% Nk603 1% Bt11 1%
E 0.5% 0.031 % Mon810 0.1% Nk603 0.1% Bt11 0.1%
F 0.5% 0.031 % Mon810 0.1% Nk603 0.1% Bt11 0.1%
G 0.25% 0.015 % Chalqueño Control Negativo MEB
H 0.25% 0.015 % Chalqueño Control Negativo MEB
1 2 3 4 5
A
B
C
D
E
F
G
H
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Detection thresholds of three transgenic maize events
CP4-EPSPS protein can be detected with high sensitivity in mixtures with low percentage of transgenic maize.
Sensitivity for detection of CRY proteins is much lower.
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Considerations:Immunochemical methods
• Immunochemical methods can be realized in fast ways, in situ, or in laboratory, few equipment is required.
• Strip based methods are only qualitative.
• ELISA can be quantitative but:– Different levels of protein expression are reflected in
different sensitivity levels– No reference materials recognized.– No agreement between quantification units (% in
weight or protein concentration)
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Double chain opening (~90°C) –denaturation- Primers that recognize specific sequences
(50- 60°C) Synthesis of template complementary
chains (74°C)
Sequence of interest
Synthesis of one copy Taq polymerase
PCR principles
5’ 3’
3’ 5’
5’
5’3’
3’
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Sequence of interest
Amplification:
Taq
Taq
Taq
Taq
Amplification
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Number of DNA molecules124816326412825651210241,000,0001,000,000,0001,000,000,000,000
Number of PCR cycles012345678910203040
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Cycle number
PCR products
Theoretical
Real
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Factors to consider
• Specificity – primer design
• Product length (DNA amplified fragment)
• There are differences between qualitative and quantitative tests
• Whether PCR is uniplex or multiplex
• If the method is specific for a type of instrument
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1. Exploration
2. Gene specific target
3. Specific construct
4. Event specific target
LOW
HIGH
Tar
get
sp
ecif
icit
y
HH Host genomic DNAPP Promoter element (CaMV 35S)EE Amplifying elementGG Gene of interest (Cry, EPSPS) TT Terminator (NOS)
PP EE HHHH GG TT
Primer design
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Recombinant Gene
A B C D
1 2 3 4 5
Amplicons
PCR for transgenic PCR for transgenic sequence detectionsequence detection
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Recombinant gene
Genomic DNA
DNA of GM grainsDNA of GM grains
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Specific-specie primers recognize
genomic DNA
Genomic DNA
Specific-GMO primers do not
interact
DNA of non-GM DNA of non-GM grainsgrains
No amplification
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Intrinsic factors of the sample that affect the amplification
• Integrity of template DNA– Size of the amplicon
• Presence of inhibitory substances– Humic substances– Proteins
Others:– EDTA– NaOH– SDS and other detergents
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1 Ladder 2A Seeds
2B Commercial nixtamal flour
3 Dough
4 Nixtamal flour
5 Tortilla6 Tortilla chips7 Corn chips8 Dry Corn chips
Load: 100ng
Dye: SYBR Green
2000 bp
1000 bp
1500 bp
500 bp
A
B
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
2000 bp
1000 bp
1500 bp
500 bp
A
B
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
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DNA Extraction
• Extraction yield– Tissue
• DNA purity
• Quality for amplification
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Amplification – effect of the purity of the DNA template
M S
225pb
E HJ HJS HM HMS Es P C-A
C-
225pb
M HM HMS HJ HJS S E Es P
B
225pb
C- M HM HMS HJ HJS S E PC
Amplification of a fragment of theInvertase gene.
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Limit of Detection
300 pb
10 1 0.1M
MON810
BC-1 C-4C-2 C-3
100 pb
Detection of the specific event MON810 in different proportions (10%, 1% y 0.1%). C-1, negative control with BT11 maize seed DNA 100% transgenic; C-2, negative control with NK603 maize seed genomic DNA; C-3, negative control with chalqueño maize seed DNA; C-4, negative control without DNA. M, 50 bp ladder.
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Amplicons of CamV35S promoter and restriction products with Asp700. Lane 1: 50bp ladder, lanes 2 and 3: Bt176 control, lanes 4 and 5: canned corn grains, in 2% agarose gel.
Identity verification:Restriction analysis
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Quantitative PCR qPCR – RTQ-PCR
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10 2 1 0.1100 0.01
Cycle number
PCRproducts
Ct
Log conc.
m=-3.32 Ct = number of cycles needed for the amplification signal tobe statistically different from the background signal
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Number of DNA molecules
2 2 1
4 2 2
8 2 3
10 2 3.32
16 2 4
32 ...64 ...128 ...
Number of PCR cycles
123
4567
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Lineal dynamic range
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Efficiency
Efficiency = ([10 ]-1) 100(-1/m)*
Results are accepted when the efficiencyis higher than 95% (m = 3.45 a 3.3)
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More common system probes
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Some results in PCR real time
Maíze %GMO
Primers and ProbesBt11
Primers and ProbeMON810
Primers and ProbePromoter 35S CaMV
Primers and ProbeEndogenous gene
No. muestras/reacciones positivas
Promedio
de Ct
No. muestras/reacciones positivas
Promedio de Ct
No. muestras/reacciones positivas
Promedio de Ct
No. muestras/reacciones positivas
Promedio de Ct
Bt11 10 3/3 29.2+0.1 0/3 - 8/8 26.1+0.2 10/10 23+0.33
MON810 10 0/3 37.9 * 6/6 27.4+0.1 5/5 26.4+0.1 10/10 22.9+0.5
NK603 10 0/3 39.8+0.5*
0/3 - 4/4 25.7+0.2 6/6 23+0.16
Chalqueño 0 0/3 - 0/3 - 0/6 35.9+0.3* 10/10 23+0.42
NTC - 0/3 - 0/3 - 0/4 - 0/10 -
Specificity of primers and probes designed for RTQ-PCR
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Effect of the extraction system
Figure 26. Standard curves of the events MON810 y Bt11 from DNA extracted with the commercial systems A, B and C. 1, curve of the event Bt11 with extraction system A. 2, curve of the event MON810 with extraction system A. 3, curve of the event MON810 with extraction system B. 4, curve of the event Bt11 with extraction system B. 5, curve of the event Bt11 with extraction system C. 6, curve of the event MON810 with extraction system C.
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Figure 27. Amplification curves generated with 100% transgenic DNA from event MON810, extracted with the system B. A, 20 ng. B, 10 ng. C, 5 ng. D, 2.5 ng. E, 1.25 ng. F, 0.625 ng. G, 0.313 ng. H, 0.156 ng.
A B C D E F G H
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Figure 29. Amplification curves generated with 100% transgenic DNA of the event MON810, extracted with the system A. Serial dilutions were performed however, the amplification generated with each dilution does not allow to clearly establish to each curve the initial DNA concentration.
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Effect of the extraction method over the quantification
Mixture of MON810 (%)
10 1 0.1
Extraction method
B 11.48 + 0.327 0.91 + 0.186 0.11 + 0.032
C16.15 + 1.118 2.11 + 0.154 0.12 + 0.016
A 13.96 + 0.140 Without amplification
Without amplification
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Effect of the primers and probes design
Linearization Data
Curve with event
Slope Intercept R2 Efficiency (%)
Transgene
Kit comercial
OGMs
Bt11 -3.537 28.253 0.999 91.7
MON810 -3.375 29.712 0.999 97.8
NK603 -3.836 30.512 0.999 82.3
Diseño
Bt11 -3.382 27.673 0.998 97.5
MON810 -3.326 28.411 0.998 99.8
NK603 -3.429 29.592 0.992 95.7
Endogenous Gene
Kit comercial
OGMs
Bt11 -3.685 29.003 0.993 86.8
MON810 -3.516 29.466 0.996 92.5
NK603 -3.731 30.187 0.985 85.4
Diseño
Bt11 -3.111 28.593 0.997 109.6
MON810 -3.326 28.228 0.995 99.8
NK603 -3.193 29.103 0.995 105.7
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Sybr green
First negative derivate of thedissociation curves of Amplicons obtained for A.MON810 andB. endogenous gene
A
B
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Non-specific amplification
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Mixture of MON810 (%)
10 1 0.1
Extraction method
B 10.3±0.007 1.1±0.021 0.25±0.006
C 10.8±0.018 2.3±0.017 0.10±0.077
A 48.5±0.157 53.8±0.149 0.03±0.011
Effect of the DNA quality over Sybr green quantification
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Other example:
• Detection of transgenic maize in processed foods:– Nixtamal Flour– Dough– Tortilla– Fried tortilla (tostada)– Fried dough
M. Quirasco, B. Schoel, J. Plasencia, J. Fagan & A. Gálvez. 2004. Suitability of RTQ-PCR and ELISA for Cry9C detection in Mexican corn tortillas: fate of DNA and protein after alkaline cooking. Journal of AOAC International. 87:639-646
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1 Ladder 2A Semillas
2B Harina de nixtamal comercial3 Masa4 Harina de nixtamal
5 Tortilla6 Tortilla frita7 Masa frita8 Masa seca frita
Carga: 100ng
Tinción: SYBR Green
2000 bp
1000 bp
1500 bp
500 bp
A
B
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
2000 bp
1000 bp
1500 bp
500 bp
A
B
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
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Content of GMO, determined by RTQ-PCR, in different nixtamalized products prepared with white maize non – transgenic and different percentages of StarLinkTM .
Granos de maíz
0.19 0.018 9.3
Masa 0.10 0.005 4.8
Harina de nixtamal
0.10 0.001 0.7
Tortilla 0.31 0.004 1.4
Tortilla chip
ND - -
Corn chip 0.10 0.002 1.5
StarLinkTM 0.1% (w/w)
Sample Media, % (w/w)
RSD CV, %
Corn chip secos
ND - -
ND = No detectado
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Granos de maíz
1.23 0.121 9.8
Masa 1.16 0.002 0.2
Harina de nixtamal
1.03 0.016 1.5
Tortilla 1.41 0.015 1.0
Tortilla chip
0.52 0.001 0.3
Corn chip 1.16 0.023 2.0
StarLinkTM 1% (w/w)
Sample Media , % (w/w)
RSD CV, %
Corn chip secos
0.63 0.079 12.4
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Granos de maíz
14.12 0.166 1.2
Masa 12.64 0.090 0.7
Harina de nixtamal
9.35 0.017 0.2
Tortilla 9.47 0.254 2.7
Tortilla chip
6.64 0.175 2.6
Corn chip 14.29 0.119 0.8
StarLinkTM 10% (w/w)
Sample Media, % (w/w)
RSD CV, %
Corn chip secos
8.28 0.053 0.6
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LOD = Limit of Detection=.01% en RTq-PCR
LOQ = Limit of Quantification= 0.1% en RTq-PCR
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Immunochemical methods are adequate to detect the protein in grains and materials without too much processing
Seed Primary Processed Highly processed
ingredient foods foods
Protein
DNA
... However DNA can be detected in highly processed foods
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ELISAELISA
ImmunostripImmunostripss
p35Sp35S
EventEventSpecificSpecific
Composite Composite samples samples
““
Pre-treatmentPre-treatment
Halving 1Halving 1Halving 2Halving 2
Detection of Detection of authorized and authorized and unauthorized unauthorized
transgenic events in transgenic events in Mexico by RTQ-PCRMexico by RTQ-PCR
HeterologouHeterologous protein s protein detectiondetection
Detection and Detection and cuantification of cuantification of
exogenous DNA by exogenous DNA by RTQ-PCRRTQ-PCR
SamplingSampling
GenScanGenScan
ResultsResults
UNAMUNAM
11 22 33
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Piedras NegrasPiedras Negras
Cd. JuárezCd. Juárez
MatamorosMatamoros
AltamiraAltamira
Veracruz CentroVeracruz CentroVeracruz 430Veracruz 430
CoatzacoalcosCoatzacoalcos
Nuevo LaredoNuevo Laredo
Customs wheremaize sampleswere obtained
Customs wheremaize sampleswere obtained
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Subsampling by dividing into four parts (“halving”)Subsampling by dividing into four parts (“halving”)
1)1)
2)2)
3)3)
4)4)
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Food authorization status of GM varieties of maize
TRANSFORMATION EVENTAuthorized in
Mexico U.S.A. Europe
MON-00021-9 (GA21) Yes Yes Yes
MON-00603-6 (NK603) Yes Yes Yes
MON-00810-6 (MON810) Yes Yes Yes
DAS-01507-1 (TC1507) Yes Yes Yes
MON-00863-5 (MON863) Yes Yes Yes
DAS-59122-7 Yes Yes Yes
MON-88017-3 (MON88017) Yes Yes No
ACS–ZM002-1 / ACS-ZM003-2 (T14, T25) Yes Yes Yes
SYN-BTØ11-1 (BT11 (X4334CBR, X4734CBR)) Yes * Yes Yes
REN-00038-3 (LY038) Yes * Yes No
SYN-IR604-5 (MIR604) Yes * Yes No
* vents Approved in october of 2007* vents Approved in october of 2007
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Immunological methods commercially available for heterologous protein
detection
Immunological methods commercially available for heterologous protein
detectionELISA• CP4-EPSPS (RR)• Cry3Bb1• Cry1Ab/1Ac• Cry1F
ELISA• CP4-EPSPS (RR)• Cry3Bb1• Cry1Ab/1Ac• Cry1F
Immunostrips• CP4-EPSPS (RR)• Cry3Bb1• Cry1Ab/1Ac• Cry1F• Cry34Ab1• Cry9C• PAT
Immunostrips• CP4-EPSPS (RR)• Cry3Bb1• Cry1Ab/1Ac• Cry1F• Cry34Ab1• Cry9C• PAT
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Possible presence of transgenic events by immunoassay (ELISA + strips)
Possible presence of transgenic events by immunoassay (ELISA + strips)
Protein Port 1 Port 2 Port 3 Poprt 4 Port 5 Port 6 Port 7 Port 8
Cry1Ab/Ac
MON810Bt11
MON80100*MON802*MON809*
MON810Bt11
MON80100*MON802*MON809*
MON810Bt11
MON80100*MON802*MON809*DBT418**
MON810Bt11
MON80100*MON802*MON809*
MON810Bt11
MON80100*MON802*MON809*DBT418**
MON810Bt11
MON80100*MON802*MON809*DBT418 **
MON810Bt11
MON80100*MON802*MON809*
MON810Bt11
MON80100*MON802*MON809*
Cry3Bb1
MON88017MON863
MON88017MON863
MON88017MON863
MON88017MON863
MON88017MON863
MON88017MON863
MON88017MON863 N/D
Cry1F DAS01507 DAS01507 DAS01507DAS06275 *** DAS01507 DAS01507
DAS06275***DAS01507
DAS06275 *** DAS01507 DAS01507
Cry34Ab1
DAS59122 DAS59122 DAS59122 DAS59122 DAS59122 DAS59122 DAS59122
Cry9C ND ND ND ND ND ND ND ND
CP4-EPSPS
NK603MON88017MON80100*
MON802*MON809*
NK603MON88017MON80100*
MON802*MON809*
NK603MON88017MON80100*
MON802*MON809*
NK603MON88017MON80100*
MON802*MON809*
NK603MON88017MON80100*
MON802*MON809*
NK603MON88017MON80100*
MON802*MON809*
NK603MON88017MON80100*
MON802*MON809*
NK603MON88017MON80100*
MON802*MON809*
PAT† ND ND
MON863DAS59122T14/T25
DAS06275***DBT418**
MS6
ND
MON863DAS59122T14/T25
DAS-06275***DBT418**
MS6
MON863DAS59122T14/T25
DAS06275***DBT418**
MS6
ND ND
ND, non detected protein* Events not containing Cry1Ab/Ac + CP4EPSPS** Event containing Cry1Ab/Ac + PAT*** Event containing Cry1F + PAT
ND, non detected protein* Events not containing Cry1Ab/Ac + CP4EPSPS** Event containing Cry1Ab/Ac + PAT*** Event containing Cry1F + PAT
† StarLink contains Cry9C + PAT. Cry9C not detected, its presence is discarded in PAT positive samples
Potential presence of MON80100 y MON809 because they were not commercialized. DNA verification required.
† StarLink contains Cry9C + PAT. Cry9C not detected, its presence is discarded in PAT positive samples
Potential presence of MON80100 y MON809 because they were not commercialized. DNA verification required.
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EventEvent
MON810MON810 T25T25 GA21GA21 NK603NK603 DAS-DAS-
01507-101507-1
MON863MON863 MON8801MON8801
77
DAS-DAS-59122-759122-7
11 22 33 44 55 66 77 88
Specific presence of transgenic events in maize grains, by RTQ-PCR
Specific presence of transgenic events in maize grains, by RTQ-PCR
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Third party analysis (Gene Scan)Third party analysis (Gene Scan)
• Confirmed the results obtained in Lab. 312, Dept. of Food Science and Biotecnhnology, Faculty of Chemistry, UNAM
• Confirmed the absence of the eventMON88017
• Confirmed the results obtained in Lab. 312, Dept. of Food Science and Biotecnhnology, Faculty of Chemistry, UNAM
• Confirmed the absence of the eventMON88017
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“Possible” events are discarded“Possible” events are discarded
• Do not encompass the totality of authorized events (GA21), as well as non-authorized ones (676/678/680, DLL25, LY038, MIR604, MS3 y MS6)
• Do not encompass the totality of authorized events (GA21), as well as non-authorized ones (676/678/680, DLL25, LY038, MIR604, MS3 y MS6)
• Immunodetection is insufficient for specific-event detection.
• Immunodetection is insufficient for specific-event detection.
• By PCR was confirmed the presence of MON810, MON862, DAS01507, DAS59122 and NK603
• It was also discarded the presence of MON88017, possible event according to the immunodetection.
• By PCR was confirmed the presence of MON810, MON862, DAS01507, DAS59122 and NK603
• It was also discarded the presence of MON88017, possible event according to the immunodetection.
• Technological development is needed for the detection of possible unauthorized events.
• Technological development is needed for the detection of possible unauthorized events.
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Quantification (RTQ-PCR) of transgenic events authorized in Mexico prior to may 2007*
* Bt11 y LY038 approved in july andy MIR604 in october of 2007
Quantification (RTQ-PCR) of transgenic events authorized in Mexico prior to may 2007*
* Bt11 y LY038 approved in july andy MIR604 in october of 2007
%
CustomsEvents
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Total content (%) of GM material in each one of the analyzed samples
Total content (%) of GM material in each one of the analyzed samples
%
Aduana
Estado
Región
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Event 1 x Event 2 x Event 3ACS-ZMØØ3-2 x MON-ØØ81Ø-
6
DAS-59122-7 x NK603
DAS-59122-7 x TC1507 x NK603
DAS-Ø15Ø7-1 x MON-ØØ6Ø3-6
BT11 x MIR604
BT11 x MIR604 x GA21
MON-ØØ6Ø3-6
x MON-ØØ81Ø-6
MON-ØØ81Ø-6
x LY038
MON-ØØ863-5
x MON-ØØ6Ø3-6
MON-ØØ863-5
x MON-ØØ81Ø-6
MON-ØØ863-5
x MON-ØØ81Ø-6
x MON-ØØ6Ø3-6
MON-ØØØ21-9
x MON-ØØ81Ø-6
MON89034 x MON88017
MON8903 x NK603
SYN-BTØ11-1 x MON-ØØØ21-9
TC1507 x DAS-59122-7
MIR604 x GA21
MON810 x MON88017
“Stacked” varieties of Maize“Stacked” varieties of Maize
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Evento “Stack” DAS59122-7 x NK603
Copia 1 ADN
Copia 2 ADN
Copia 3 ADN
Copia 4 ADN
Copia 5 ADN 100% 100%
200% ?
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Evento “Stack” DAS59122-7 x NK603
Copia 1 ADN
Copia 2 ADN
Copia 3 ADN
Copia 4 ADN
Copia 5 ADN 80% 100%
180% ?
180% ?
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Evento “Stack” DAS59122-7 x NK603
Copia 1 ADN(No-GM)
Copia 2 ADN
Copia 3 ADN
Copia 4 ADN
Copia 5 ADN
80% 80%
160% ?
160% ?
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Normalizing genes from developers´ certified methods*
Normalizing genes from developers´ certified methods*
• MON810: hmg (high mobility group)• MON863: adh (alcohol
dehydrogenase)• NK603: adh (alcohol dehydrogenase)
• MON810: hmg (high mobility group)• MON863: adh (alcohol
dehydrogenase)• NK603: adh (alcohol dehydrogenase)
* Only recomended gene
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Use of normalizing gene in tests performed in the laboratory: RT-PCR
Use of normalizing gene in tests performed in the laboratory: RT-PCR
• MON810 with hmg
15.25 x 100 = 43.834.84
• MON810 with hmg
15.25 x 100 = 43.834.84
• MON810 with adh
15.25 x 100 = 23.166.01
• MON810 with adh
15.25 x 100 = 23.166.01
• % OGM = (transgene/normalizing gene) x 100
• % OGM = (transgene/normalizing gene) x 100
• If the amount of endogenous gene increases (denominator), the amount of GMO will be underestimated.
• If the amount of endogenous gene increases (denominator), the amount of GMO will be underestimated.
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%
Quantification of three events using
two different normalizing
Quantification of three events using
two different normalizing
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• adh could be present in the genome of the maize in more than one copy.
• Quantification with adh could underestimate the results
• adh could be present in the genome of the maize in more than one copy.
• Quantification with adh could underestimate the results
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Quantification of GM material (total) according to specific events
or promoter 35S
Quantification of GM material (total) according to specific events
or promoter 35S
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Hipotesis about the diferent quantification
of the endogenous gene
Hipotesis about the diferent quantification
of the endogenous gene• Poor characterization of the endogenous
gene (number of copies in the genomic DNA not specified)
• Sequences of primers or probes provided by the developer are not specific enough
• Low astringency PCR programme• ¿Which endogenous gene should choose? • ¿Differences between cultivars?
• Poor characterization of the endogenous gene (number of copies in the genomic DNA not specified)
• Sequences of primers or probes provided by the developer are not specific enough
• Low astringency PCR programme• ¿Which endogenous gene should choose? • ¿Differences between cultivars?
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ConclusionsConclusions• Exists a high income of GM material in the
analyzed customs.• The most abundant events were: MON810 >
NK603 > DAS1507-1• The presence of “stacked” variety makes more
complex the transgenic material quantification.• Standardization is required (at international
level) regarding which normalizing genes should be used.
• An inter- governmental information exchange system is required in order to harmonize maize commerce between the USA and Mexico
• The event MON88017 (authorized variety) was not detected
• Exists a high income of GM material in the analyzed customs.
• The most abundant events were: MON810 > NK603 > DAS1507-1
• The presence of “stacked” variety makes more complex the transgenic material quantification.
• Standardization is required (at international level) regarding which normalizing genes should be used.
• An inter- governmental information exchange system is required in order to harmonize maize commerce between the USA and Mexico
• The event MON88017 (authorized variety) was not detected
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Abraham Acatzi1
Javier Magaña1
Carlos Moles2
Carolina Peña2
Marcela Castillo2
Maricarmen Quirasco1
Javier Plasencia3 Marcelo Signorini4
Amanda Gálvez1, 2
1 Programa Universitario de Alimentos. PUAL-UNAM2 Dept. Alimentos y Biotecnología. Facultad de Química. Universidad Nacional Autónoma de México (UNAM)3 Dept. Bioquímica. Facultad de Química. UNAM4 Comisión Federal para la Protección contra Riesgos Sanitarios. Secretaría de SALUD