item 7. sampling and lmos detection 7.3. identification of lmos

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• For environmental risk verifications

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

• 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

Sampling

Extraction of:

Protein

DNA

PCR end point

RTq-PCR

Immunostrips

ELISA plates

In order to detect proteins, specific antibodies are required. The antibodies are proteins with quaternary structure.

Control line. Antibodies anti-IgG adsorbed

Test line. Antibodies anti-antigen adsorbedAntibodies anti-antigen

conjugated with enzyme

Lateral flow test, IMMUNOSTRIP

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

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

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

Qualitative immunoassay in strip.

Ej. Cry9C QuickStixTM Envirologix

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

%

The first Ac is adsorbed to the plate

ELISA:

Enzyme-Linked-ImmunoSorbent Assay

Format: DAS (Double Antibody Sandwich)

Sample that contains protein is added

Protein

ELISA:

Enzyme-Linked-ImmunoSorbent Assay

Formato: DAS (Double Antibody Sandwich)

Second antibody is added. It is conjugated with a enzyme

Enzyme

Antibody

ELISA:

Enzyme-Linked-ImmunoSorbent Assay

Format: DAS (Double Antibody Sandwich)

Colorless

compound

Colorless

compoundProduct Product

Enzyme

Antibody

ELISA:

Enzyme-Linked-ImmunoSorbent Assay

Formato: DAS (Double Antibody Sandwich)

ELISA plate, second antibody congujated to alkaline phospatase

Quantitative Immunoassay (ELISA)

Sample extraction.Centrifugation to clarify samples

Preparation of ELISA plates

Sample applicationIncubation at room temperature

Wash

Substrate addition/ Colour development Spectrophotometric

measurement - for quantification-

ELISA cont.

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

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

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.

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)

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’

Sequence of interest

Amplification:

Taq

Taq

Taq

Taq

Amplification

Number of DNA molecules124816326412825651210241,000,0001,000,000,0001,000,000,000,000

Number of PCR cycles012345678910203040

Cycle number

PCR products

Theoretical

Real

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

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

Recombinant Gene

A B C D

1 2 3 4 5

Amplicons

PCR for transgenic PCR for transgenic sequence detectionsequence detection

Recombinant gene

Genomic DNA

DNA of GM grainsDNA of GM grains

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

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

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

DNA Extraction

• Extraction yield– Tissue

• DNA purity

• Quality for amplification

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.

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.

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

Quantitative PCR qPCR – RTQ-PCR

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

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

Lineal dynamic range

Efficiency

Efficiency = ([10 ]-1) 100(-1/m)*

Results are accepted when the efficiencyis higher than 95% (m = 3.45 a 3.3)

More common system probes

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

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.

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

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.

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

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

Sybr green

First negative derivate of thedissociation curves of Amplicons obtained for A.MON810 andB. endogenous gene

A

B

Non-specific amplification

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

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

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

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

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

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

LOD = Limit of Detection=.01% en RTq-PCR

LOQ = Limit of Quantification= 0.1% en RTq-PCR

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

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

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

Subsampling by dividing into four parts (“halving”)Subsampling by dividing into four parts (“halving”)

1)1)

2)2)

3)3)

4)4)

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

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

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.

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

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

“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.

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

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

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

Evento “Stack” DAS59122-7 x NK603

Copia 1 ADN

Copia 2 ADN

Copia 3 ADN

Copia 4 ADN

Copia 5 ADN 100% 100%

200% ?

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% ?

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% ?

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

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.

%

Quantification of three events using

two different normalizing

Quantification of three events using

two different normalizing

• 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

Quantification of GM material (total) according to specific events

or promoter 35S

Quantification of GM material (total) according to specific events

or promoter 35S

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?

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

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