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Item 7. SAMPLING AND LMOs DETECTION 7.3. IDENTIFICATION OF LMOs.

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Presentation on theme: "Item 7. SAMPLING AND LMOs DETECTION 7.3. IDENTIFICATION OF LMOs."— Presentation transcript:

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2 Item 7. SAMPLING AND LMOs DETECTION 7.3. IDENTIFICATION OF LMOs

3 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

4 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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6 First GM-food authorized 1994 First 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 market The variety lost sensory characteristics and was retired from the market Historical cans of GM tomato puree with long shelf life Historical cans of GM tomato puree with long shelf life

7 Sampling Extraction of: Protein DNA PCR end point RTq-PCR Immunostrips ELISA plates

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

9 Control line. Antibodies anti- IgG adsorbed Test line. Antibodies anti- antigen adsorbed Antibodies anti-antigen conjugated with enzyme Lateral flow test, IMMUNOSTRIP

10 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

11 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

12 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

13 Qualitative immunoassay in strip. Ej. Cry9C QuickStix TM Envirologix

14 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 %

15 The first Ac is adsorbed to the plate ELISA: Enzyme-Linked-ImmunoSorbent Assay Format: DAS (Double Antibody Sandwich)

16 Sample that contains protein is added Protein ELISA: Enzyme-Linked-ImmunoSorbent Assay Formato: DAS (Double Antibody Sandwich)

17 Second antibody is added. It is conjugated with a enzyme Enzyme Antibody ELISA: Enzyme-Linked-ImmunoSorbent Assay Format: DAS (Double Antibody Sandwich)

18 Colorless compound Colorless compound Product Enzyme Antibody ELISA: Enzyme-Linked-ImmunoSorbent Assay Formato: DAS (Double Antibody Sandwich)

19 ELISA plate, second antibody congujated to alkaline phospatase

20 Quantitative Immunoassay (ELISA) Sample extraction. Centrifugation to clarify samples Preparation of ELISA plates

21 Sample application Incubation at room temperature Wash Substrate addition/ Colour development Spectrophotometric measurement - for quantification- ELISA cont.

22 Specificity assesment of antibodies in the ELISA test ABCDEFGHABCDEFGH ABCDEFGHABCDEFGH 123 A MEBCry 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 EPSPS KIT Cry 1Ab/1Ac

23 Quantitative Analysis by ELISA in detection of protein CP4-EPSPS A MEB % Mon810 10%Nk603 10% Bt11 10% B MEB %Mon810 10%Nk603 10% Bt11 10% C 1% %Mon810 1%Nk603 1%Bt11 1% D 1% %Mon810 1%Nk603 1%Bt11 1% E 0.5% %Mon %Nk %Bt11 0.1% F 0.5% %Mon %Nk %Bt11 0.1% G 0.25% %Chalqueño Control Negativo MEB H 0.25% %Chalqueño Control Negativo MEB ABCDEFGHABCDEFGH

24 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.

25 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)

26 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

27 Sequence of interest Amplification: Taq Amplification

28 Number of DNA molecules ,000,000 1,000,000,000 1,000,000,000,000 Number of PCR cycles

29 Cycle number PCR products Theoretical Real

30 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

31 1. Exploration 2. Gene specific target 3. Specific construct 4. Event specific target LOW HIGH Target specificity H H Host genomic DNA P P Promoter element (CaMV 35S) E E Amplifying element G G Gene of interest (Cry, EPSPS) T T Terminator (NOS) PE H HG T Primer design

32 Recombinant Gene ABCD Amplicons PCR for transgenic sequence detection

33 Recombinant gene Genomic DNA DNA of GM grains

34 Specific-specie primers recognize genomic DNA Genomic DNA Specific-GMO primers do not interact DNA of non-GM grains No amplification

35 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

36 1 Ladder 2A Seeds 2B Commercial nixtamal flour 3 Dough 4 Nixtamal flour 5 Tortilla 6 Tortilla chips 7 Corn chips 8 Dry Corn chips Load: 100ng Dye: SYBR Green

37 DNA Extraction Extraction yield –Tissue DNA purity Quality for amplification

38 Amplification – effect of the purity of the DNA template M S 225 pb E HJ HJSHM HMS Es P C- A 225 pb M HM HMS HJ HJS S E EsP B 225 pb C- MHMHMS HJ HJS S E P C Amplification of a fragment of the Invertase gene.

39 Limit of Detection 300 pb M MON810 B C-1 C-4 C-2 C 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.

40 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

41 Quantitative PCR qPCR – RTQ-PCR

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43 Cycle number PCR products Ct Log conc. m=-3.32 Ct = number of cycles needed for the amplification signal to be statistically different from the background signal

44 Number of DNA molecules Number of PCR cycles

45 Lineal dynamic range

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47 Efficiency Efficiency = ([10 ]-1) 100 (-1/m) * Results are accepted when the efficiency is higher than 95% (m = 3.45 a 3.3)

48 More common system probes

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50 Some results in PCR real time Maíze% GM O Primers and Probes Bt11 Primers and Probe MON810 Primers and Probe Promoter 35S CaMV Primers and Probe Endogenous gene No. muestras/ reacciones positivas Promedi o de Ct No. muestras/ reacciones positivas Promedio de Ct No. muestras/ reacciones positivas Promedio de Ct No. muestras/ reacciones positivas Promedio de Ct Bt11103/ /3-8/ / MON810100/337.9 *6/ / / NK603100/ * 0/3-4/ / Chalqueño00/3- -0/ *10/ NTC-0/3- -0/4-0/10- Specificity of primers and probes designed for RTQ-PCR

51 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.

52 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, ng. G, ng. H, ng. A B C D E F G H

53 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.

54 Effect of the extraction method over the quantification Mixture of MON810 (%) Extraction method B C A Without amplification

55 Effect of the primers and probes design Linearization Data Curve with event SlopeInterceptR2R2 Efficiency (%) Transgene Kit comercial OGMs Bt MON NK Diseño Bt MON NK Endogenous Gene Kit comercial OGMs Bt MON NK Diseño Bt MON NK

56 Sybr green First negative derivate of the dissociation curves of Amplicons obtained for A.MON810 and B. endogenous gene A B

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58 Non-specific amplification

59 Mixture of MON810 (%) Extraction method B 10.3± ± ±0.006 C 10.8± ± ±0.077 A 48.5± ± ±0.011 Effect of the DNA quality over Sybr green quantification

60 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 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:

61 1 Ladder 2A Semillas 2B Harina de nixtamal comercial 3 Masa 4 Harina de nixtamal 5 Tortilla 6 Tortilla frita 7 Masa frita 8 Masa seca frita Carga: 100ng Tinción: SYBR Green

62 Content of GMO, determined by RTQ-PCR, in different nixtamalized products prepared with white maize non – transgenic and different percentages of StarLink TM. Granos de maíz Masa Harina de nixtamal Tortilla Tortilla chip ND-- Corn chip StarLink TM 0.1% (w/w) SampleMedia, % (w/w) RSDCV, % Corn chip secos ND-- ND = No detectado

63 Granos de maíz Masa Harina de nixtamal Tortilla Tortilla chip Corn chip StarLink TM 1% (w/w) SampleMedia, % (w/w) RSD CV, % Corn chip secos

64 Granos de maíz Masa Harina de nixtamal Tortilla Tortilla chip Corn chip StarLink TM 10% (w/w) SampleMedia, % (w/w) RSD CV, % Corn chip secos

65 LOD = Limit of Detection =.01% en RTq-PCR LOQ = Limit of Quantification = 0.1% en RTq-PCR

66 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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68 ELISA Immunostrips p35S EventSpecific Composite samples Pre-treatment Halving 1 Halving 2 Detection of authorized and unauthorized transgenic events in Mexico by RTQ-PCR Heterologous protein detection Detection and cuantification of exogenous DNA by RTQ-PCR Sampling GenScan Results UNAM123

69 Piedras Negras Cd. Juárez Matamoros Altamira Veracruz Centro Veracruz 430 Coatzacoalcos Nuevo Laredo Customs where maize samples were obtained Customs where maize samples were obtained

70 Subsampling by dividing into four parts (halving) 1) 2) 3) 4)

71 Food authorization status of GM varieties of maize TRANSFORMATION EVENT Authorized in Mexico U.S.A.Europe MON (GA21)Yes MON (NK603)Yes MON (MON810)Yes DAS (TC1507)Yes MON (MON863)Yes DAS Yes MON (MON88017)Yes No ACS–ZM002-1 / ACS-ZM003-2 (T14, T25)Yes SYN-BTØ11-1 (BT11 (X4334CBR, X4734CBR))Yes *Yes REN (LY038)Yes *YesNo SYN-IR604-5 (MIR604)Yes *YesNo * vents Approved in october of 2007

72 Immunological methods commercially available for heterologous protein detection ELISA 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

73 Possible presence of transgenic events by immunoassay (ELISA + strips) ProteinPort 1Port 2Port 3Poprt 4Port 5Port 6Port 7Port 8 Cry1Ab/Ac MON810 Bt11 MON80100* MON802* MON809* MON810 Bt11 MON80100* MON802* MON809* MON810 Bt11 MON80100* MON802* MON809* DBT418** MON810 Bt11 MON80100* MON802* MON809* MON810 Bt11 MON80100* MON802* MON809* DBT418** MON810 Bt11 MON80100* MON802* MON809* DBT418 ** MON810 Bt11 MON80100* MON802* MON809* MON810 Bt11 MON80100* MON802* MON809* Cry3Bb1 MON88017 MON863 MON88017 MON863 MON88017 MON863 MON88017 MON863 MON88017 MON863 MON88017 MON863 MON88017 MON863 N/D Cry1F DAS01507 DAS06275 *** DAS01507 DAS06275*** DAS01507 DAS06275 *** DAS01507 Cry34Ab1 DAS59122 Cry9C ND CP4- EPSP S NK603 MON88017 MON80100* MON802* MON809* NK603 MON88017 MON80100* MON802* MON809* NK603 MON88017 MON80100* MON802* MON809* NK603 MON88017 MON80100* MON802* MON809* NK603 MON88017 MON80100* MON802* MON809* NK603 MON88017 MON80100* MON802* MON809* NK603 MON88017 MON80100* MON802* MON809* NK603 MON88017 MON80100* MON802* MON809* PAT ND MON863 DAS59122 T14/T25 DAS06275*** DBT418** MS6 ND MON863 DAS59122 T14/T25 DAS-06275*** DBT418** MS6 MON863 DAS59122 T14/T25 DAS06275*** DBT418** MS6 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.

74 Event MON810 T25 GA21 NK603 DAS MON863 MON88017 DAS Specific presence of transgenic events in maize grains, by RTQ-PCR

75 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

76 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) 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.

77 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 % Customs Events

78 Total content (%) of GM material in each one of the analyzed samples % AduanaEstadoRegión

79 Event 1xEvent 2xEvent 3 ACS-ZM ØØ 3-2xMON- ØØ 81 Ø -6 DAS xNK603 DAS xTC1507xNK603 DAS- Ø 15 Ø 7-1xMON- ØØ 6 Ø 3-6 BT11xMIR604 BT11xMIR604xGA21 MON- ØØ 6 Ø 3-6xMON- ØØ 81 Ø -6 xLY038 MON- ØØ 863-5xMON- ØØ 6 Ø 3-6 MON- ØØ 863-5xMON- ØØ 81 Ø -6 MON- ØØ 863-5xMON- ØØ 81 Ø -6xMON- ØØ 6 Ø 3-6 MON- ØØØ 21-9xMON- ØØ 81 Ø -6 MON89034xMON88017 MON8903xNK603 SYN-BT Ø 11-1xMON- ØØØ 21-9 TC1507xDAS MIR604xGA21 MON810xMON88017 Stacked varieties of Maize

80 Evento Stack DAS x NK603 Copia 1 ADN Copia 2 ADN Copia 3 ADN Copia 4 ADN Copia 5 ADN 100% 200% ?

81 Evento Stack DAS x NK603 Copia 1 ADN Copia 2 ADN Copia 3 ADN Copia 4 ADN Copia 5 ADN 80%100% 180% ?

82 Evento Stack DAS x NK603 Copia 1 ADN (No-GM) Copia 2 ADN Copia 3 ADN Copia 4 ADN Copia 5 ADN 80% 160% ?

83 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

84 Use of normalizing gene in tests performed in the laboratory: RT-PCR MON810 with hmg x 100 = MON810 with hmg x 100 = MON810 with adh x 100 = % OGM = (transgene/normalizing gene) x 100 If the amount of endogenous gene increases (denominator), the amount of GMO will be underestimated.

85 % Quantification of three events using two different normalizing

86 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

87 Quantification of GM material (total) according to specific events or promoter 35S

88 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?

89 Conclusions Exists a high income of GM material in the analyzed customs. The most abundant events were: MON810 > NK603 > DAS 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 > DAS 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

90 Abraham Acatzi 1 Javier Magaña 1 Carlos Moles 2 Carolina Peña 2 Marcela Castillo 2 Maricarmen Quirasco 1 Javier Plasencia 3 Marcelo Signorini 4 Amanda Gálvez 1, 2 1 Programa Universitario de Alimentos. PUAL-UNAM 2 Dept. Alimentos y Biotecnología. Facultad de Química. Universidad Nacional Autónoma de México (UNAM) 3 Dept. Bioquímica. Facultad de Química. UNAM 4 Comisión Federal para la Protección contra Riesgos Sanitarios. Secretaría de SALUD


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