Presentation on theme: "Item 7. SAMPLING AND LMOs DETECTION 7.3. IDENTIFICATION OF LMOs"— Presentation transcript:
1 Item 7. SAMPLING AND LMOs DETECTION 7.3. IDENTIFICATION OF LMOs
2 Detection is required when: By law in the country is required identification and/or labellingMixtures between GMOs + non-GMOsNeed to export to a country with strict legislationNeed to verify non-GMOs shipmentsFor environmental risk verifications
3 In the international market Important focus differences between commercial blocks:USA does not require identification and GM crops are easily set free into the environmentEU requires labelling with 0.9% treshold
5 “Historical” cans of GM tomato puree with long shelf life First GM-food authorized 1994They were labelled… and people bought them!!!The variety lost sensory characteristics and was retired from the market
6 Extraction of: Sampling Protein DNA RTq-PCR Immunostrips PCR end point ELISA platesProteinPCR end pointDNARTq-PCR
7 In order to detect proteins, specific antibodies are required In order to detect proteins, specific antibodies are required. The antibodies are proteins with quaternary structure.
8 Lateral flow test, IMMUNOSTRIP Control line. Antibodies anti-IgG adsorbedTest line. Antibodies anti-antigen adsorbedAntibodies anti-antigen conjugated with enzyme
9 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.
10 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.
11 Immunostrip procedure 1. Weigh 250 mg of fresh leaf (plantlet)2. Insert the sample in the bag3. Grind or crush the sample5. Let stand for 10 minutes (vertical position)4. Insert the immunostrip into the bag with SEB or MEB buffer.6. Read results
12 Qualitative immunoassay in strip. Ej. Cry9C QuickStixTM Envirologix
13 Also by this technique can be detected CP4-EPSPS protein in samples with low % of transgenic maize IMMUNOSTRIP Cry 1Ab/AcMaíz BT 1110%1%0.5%0.1%IMMUNOSTRIP CP4 EPSPSMaíz NK60310%1%0.1%0.05%
14 Enzyme-Linked-ImmunoSorbent Assay ELISA:Enzyme-Linked-ImmunoSorbent AssayFormat: DAS (Double Antibody Sandwich)The first Ac is adsorbed to the plate
15 Enzyme-Linked-ImmunoSorbent Assay ELISA:Enzyme-Linked-ImmunoSorbent AssayFormato: DAS (Double Antibody Sandwich)Sample that contains protein is addedProtein
16 Enzyme-Linked-ImmunoSorbent Assay ELISA:Enzyme-Linked-ImmunoSorbent AssayFormat: DAS (Double Antibody Sandwich)Second antibody is added. It is conjugated with a enzymeEnzymeAntibody
17 Enzyme-Linked-ImmunoSorbent Assay ELISA:Enzyme-Linked-ImmunoSorbent AssayFormato: DAS (Double Antibody Sandwich)ColorlesscompoundProductEnzymeAntibody
18 ELISA plate, second antibody congujated to alkaline phospatase
19 Quantitative Immunoassay (ELISA) Centrifugation to clarify samplesSample extraction.Preparation of ELISA plates
20 Incubation at room temperature Sample application ELISA cont.Incubation at room temperatureSample applicationSubstrate addition/ Colour developmentSpectrophotometric measurement for quantification-Wash
21 Specificity assesment of antibodies in the ELISA test KIT Cry 1Ab/1AcKIT CP4 EPSPSABCDEFGHABCDEFGH123AMEBCry 1Ab/1AcNK 603BCCP4Cry 3ABt 11DENPT IICry 3Bb1MON 810FGCry 2ACry 1CChalqueñoH
22 Quantitative Analysis by ELISA in detection of protein CP4-EPSPS ABCDEFGH12345AMEB0.125 %Mon %Nk603 10%Bt11 10%BMon810 10%Bt %C1%0.062 %Mon810 1%Nk603 1%Bt %DE0.5%0.031 %Mon %Nk %Bt11 0.1%FG0.25%0.015 %ChalqueñoControlNegativoHControl Negativo
23 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.
24 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 levelsNo reference materials recognized.No agreement between quantification units (% in weight or protein concentration)
25 PCR principles Double chain opening (~90°C) –denaturation- Primers that recognize specific sequences (50- 60°C)Synthesis of template complementary chains (74°C)3’ ’5’Sequence of interestSynthesis of one copyTaq polymerase3’Primers are small DNA molecules of defined sequence designed to recognize specific sequences within the genetically modified DNA present in a GMO.Once bound, primers become starting points from which Taq polymerase can synthesize a copy of a portion of the genetically modified sequences.3’5’5’ ’
26 Amplification: Amplification Sequence of interest Taq Taq Taq Taq When the Taq polymerase reaction is allowed to take place over and over again, many copies of the recombinant “target sequence” are made.Each Taq polymerase cycle doubles the number of copies.Beginning with one copy, two are made, and from that, four, and from that 8….Taq
27 Number of DNA molecules 1 2 4 8 16 32 64 128 256 512 1024 1,000,000 Number of PCR cycles12345678910203040Number of DNA molecules124816326412825651210241,000,0001,000,000,0001,000,000,000,000Continuing on, ten cycles generate more than 1000 copies of the original target sequence, 20 generate 1,000,000, thirty generate 1,000,000,000, and 40 generate 1,000,000,000,000.We normally run between 30 and 40 cycles.Obviously this is a very powerful amplification process, which is the basis for a highly sensitive detection method.
29 Factors to consider Specificity – primer design Product length (DNA amplified fragment)There are differences between qualitative and quantitative testsWhether PCR is uniplex or multiplexIf the method is specific for a type of instrument
30 Primer design P E H G T LOW HIGH 1. Exploration Target specificity1. Exploration2. Gene specific target3. Specificconstruct4. Event specific targetH Host genomic DNAP Promoter element (CaMV 35S)E Amplifying elementG Gene of interest (Cry, EPSPS)T Terminator (NOS)
31 Recombinant Gene Amplicons PCR for transgenic sequence detection A B C 12345In each case we have selected primer sets that span a unique sequence junction, that is, a site where two sequence elements have been joined in a manner unique to that event. Thus, a positive signal with these primer sets is definitive evidence for the presence of the respective event in the sample of interest.This is an example of a recombinant gene that has been created by splicing five different pieces of DNA, from five different organisms, together. These are sequences 1, 2, 3, 4, and 5. The Green bars flanking seqauences 1 and 5 represent corn genomic DNA sequences. The recombinant gene was inserted into a unique site within the corn genome, thus these are unique sequences.We show 4 different primer sets that could be used to detect this gene. Primer sets A and C detect sequences that are wholely within a single one of the five sequence elements that have been spliced together to make this recombinant gene, sequences 1, and 5, respectively. Because these two primer sets reside within a single DNA sequence element, they are unable to distinguish between the original genes, from which sequence elements 1 and 5 were obtained, and the recombinant gene, shown in the diagram. The same product will be made from these primers in either case.Primer set B, however, consists of one primer that recognizes sequence 1 and another that recognizes sequence 2. These two sequences do not exist in nature in the same gene, so the only case in which a PCR product will be made from this primer set will be when sequence elements 1 and 2 are juxtaposed, as is the case in the recombinant gene. Only then will these two sequences be juxtaposed in a manner that will allow PCR amplification to occur. Thus this primer set is specific for DNA isolated from the recombinant organism. Similarly primer set D includes one primer set specific for sequence 5 and one specific for the corn genomic sequences flanking the recombinant gene. As with primer set A, PCR products will be made from primer set D only when the recombinant gene is present, because only then will sequence 5 and the corn genomic sequences be juxtaposed. Thus primer set D is also specific for DNA isolated from the recombinant organism.Primer sets such as set B and set D both are capable of definitively identifying specific varieties of GM corn. MAFF claims to be using primer sets similar to set D. They claim that only they have access to the sequence information necessary to develop such primers. And therefore that only they can carry out definitive varietal analysis. The fact is that any properly trained molecular biologist can go into the laboratory and develop such primer sets based only on information that is in the public domain. Genetic ID has the capacity to develop primer sets such as set D and such as set B, and we use such primer sets in our varietal tests to definitively identify specific transgenic “events” or varieties, and at the same time to distinguish between DNA from transgenic corn and DNA from contaminating organisms or microorganisms.ABCDAmplicons
32 DNA of GM grains Recombinant gene Genomic DNA We use three GMO specific primers so that we have a strong internal cross-check. Information theory indicates that redundancy of information increases certainty. If we were to use only a single GMO-specific primer set, there would be no way to confirm a positive signal. If we were to use two GMO specific primer sets, we could carry out one cross-check to confirm the result. If we run three GMO specific primer sets, we can carry out three pair-wise cross checks. This increases the certainty of the result by three fold.
33 DNA of non-GM grains No amplification Specific-specie primers recognize genomic DNADNA of non-GM grainsGenomic DNASpecific-GMO primers do not interactNo amplificationResults for a non-GMO sample.
34 Intrinsic factors of the sample that affect the amplification Integrity of template DNASize of the ampliconPresence of inhibitory substancesHumic substancesProteinsOthers:EDTANaOHSDS and other detergents
36 DNA Extraction Extraction yield DNA purity Quality for amplification TissueDNA purityQuality for amplification
37 Amplification – effect of the purity of the DNA template S225pbEHJHJSHMHMSEsPC-A225pbC-MHMHMSHJHJSSEPCC-225pbMHMHMSHJHJSSEEsPBAmplification of a fragment of theInvertase gene.
38 Limit of Detection300 pb1010.1MMON810BC-1C-4C-2C-3100 pbDetection 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.
39 Identity verification: Restriction analysisAmplicons 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.
42 10210.11000.01Cycle numberPCRproductsCtCt = number of cycles neededfor the amplification signal tobe statistically different fromthe background signalm=-3.32Log conc.
43 10 2 3.32 Number of DNA molecules Number of PCR cycles 2 2 1 1 4 2 2 2 567Number of DNA molecules2 2 14 2 28 2 3Continuing on, ten cycles generate more than 1000 copies of the original target sequence, 20 generate 1,000,000, thirty generate 1,000,000,000, and 40 generate 1,000,000,000,000.We normally run between 30 and 40 cycles.Obviously this is a very powerful amplification process, which is the basis for a highly sensitive detection method.
49 Some results in PCR real time Specificity of primers and probes designed for RTQ-PCRMaíze%GMOPrimers and ProbesBt11Primers and ProbeMON810Promoter 35S CaMVEndogenous geneNo. muestras/reacciones positivasPromediode CtPromedio de Ct103/30/3-8/810/1037.9 *6/65/5NK603*4/4Chalqueño0/6*NTC0/40/10
50 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.
51 A B C D E F G HFigure 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.
52 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.
53 Effect of the extraction method over the quantification Mixture of MON810 (%)1010.1Extraction methodBCAWithout amplification
54 Effect of the primers and probes design Linearization DataCurve with eventSlopeInterceptR2Efficiency(%)TransgeneKit comercial OGMsBt11-3.53728.2530.99991.7MON810-3.37529.71297.8NK603-3.83630.51282.3Diseño-3.38227.6730.99897.5-3.32628.41199.8-3.42929.5920.99295.7EndogenousGene-3.68529.0030.99386.8-3.51629.4660.99692.5-3.73130.1870.98585.4-3.11128.5930.997109.628.2280.995-3.19329.103105.7
55 Sybr green A B First negative derivate of the dissociation curves of Amplicons obtained forMON810 andB. endogenous gene
58 Effect of the DNA quality over Sybr green quantification Mixture of MON810 (%)1010.1Extraction methodB10.3±0.0071.1±0.0210.25±0.006C10.8±0.0182.3±0.0170.10±0.077A48.5±0.15753.8±0.1490.03±0.011
59 Other example: Detection of transgenic maize in processed foods: Nixtamal FlourDoughTortillaFried tortilla (tostada)Fried doughM. 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:
60 1 Ladder2A Semillas2B Harina de nixtamal comercial3 Masa4 Harina de nixtamal5 Tortilla6 Tortilla frita7 Masa frita8 Masa seca fritaCarga: 100ngTinción: SYBR Green
61 ND StarLinkTM 0.1% (w/w) Sample Media, % (w/w) RSD CV, % Content of GMO, determined by RTQ-PCR, in different nixtamalized products prepared with white maize non – transgenic and different percentages of StarLinkTM .Granos de maíz0.190.0189.3Masa0.100.0054.8Harina de nixtamal0.0010.7Tortilla0.310.0041.4Tortilla chipND-Corn chip0.0021.5StarLinkTM 0.1% (w/w)SampleMedia, % (w/w)RSDCV, %Corn chip secosND = No detectado
62 Granos de maíz1.230.1219.8Masa1.160.0020.2Harina de nixtamal1.030.0161.5Tortilla1.410.0151.0Tortilla chip0.520.0010.3Corn chip0.0232.0StarLinkTM 1% (w/w)SampleMedia , % (w/w)RSDCV, %Corn chip secos0.630.07912.4
63 Granos de maíz14.120.1661.2Masa12.640.0900.7Harina de nixtamal9.350.0170.2Tortilla9.470.2542.7Tortilla chip6.640.1752.6Corn chip14.290.1190.8StarLinkTM 10% (w/w)SampleMedia, % (w/w)RSDCV, %Corn chip secos8.280.0530.6
64 LOD = Limit of Detection =.01% en RTq-PCRLOQ = Limit of Quantification= 0.1% en RTq-PCR
65 ... However DNA can be detected in highly processed foods Immunochemical methods are adequate to detect the protein in grains and materials without too much processingProteinDNASeed Primary Processed Highly processedingredient foods foods... However DNA can be detected in highly processed foods
67 Sampling GenScan Pre-treatment UNAM Halving 1 Halving 2 Composite samples“Detection and cuantification of exogenous DNA byRTQ-PCRHeterologous protein detection123ELISAp35SDetection of authorized and unauthorized transgenic events in Mexico by RTQ-PCREventSpecificImmunostripsResults
68 Customs where maize samples were obtained Cd. Juárez Piedras Negras Nuevo LaredoMatamorosAltamiraCustomswheremaize sampleswere obtainedVeracruz 430Veracruz CentroCoatzacoalcos
69 Subsampling by dividing into four parts (“halving”) 1)2)3)4)Subsampling by dividing into four parts (“halving”)
70 Food authorization status of GM varieties of maize TRANSFORMATION EVENTAuthorized inMexicoU.S.A.EuropeMON (GA21)YesMON (NK603)MON (MON810)DAS (TC1507)MON (MON863)DASMON (MON88017)NoACS–ZM002-1 / ACS-ZM003-2 (T14, T25)SYN-BTØ11-1 (BT11 (X4334CBR, X4734CBR))Yes *REN (LY038)SYN-IR604-5 (MIR604)* vents Approved in october of 2007
71 Immunological methods commercially available for heterologous protein detection ELISACP4-EPSPS (RR)Cry3Bb1Cry1Ab/1AcCry1FImmunostripsCP4-EPSPS (RR)Cry3Bb1Cry1Ab/1AcCry1FCry34Ab1Cry9CPAT
72 Possible presence of transgenic events by immunoassay (ELISA + strips) ProteinPort 1Port 2Port 3Poprt 4Port 5Port 6Port 7Port 8Cry1Ab/AcMON810Bt11MON80100*MON802*MON809*DBT418**DBT418 **Cry3Bb1MON88017MON863N/DCry1FDAS01507DAS06275 ***DAS06275***Cry34Ab1DAS59122Cry9CNDCP4-EPSPSNK603PAT†T14/T25MS6DAS-06275***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 samplesPotential presence of MON80100 y MON809 because they were not commercialized. DNA verification required.
73 Specific presence of transgenic events in maize grains, by RTQ-PCR MON810T25GA21NK603DASMON863MON88017DAS12345678
74 Third party analysis (Gene Scan) Confirmed the results obtained in Lab. 312, Dept. of Food Science and Biotecnhnology, Faculty of Chemistry, UNAMConfirmed the absence of the eventMON88017
75 “Possible” events are discarded Immunodetection is insufficient for specific-event detection.Do not encompass the totality of authorized events (GA21), as well as non-authorized ones (676/678/680, DLL25, LY038, MIR604, MS3 y MS6)By PCR was confirmed the presence of MON810, MON862, DAS01507, DAS59122 and NK603It was also discarded the presence of MON88017, possible event according to the immunodetection.Technological development is needed for the detection of possible unauthorized events.
76 * Bt11 y LY038 approved in july andy MIR604 in october of 2007 %EventsCustomsQuantification (RTQ-PCR) of transgenic events authorized in Mexico prior to may 2007** Bt11 y LY038 approved in july andy MIR604 in october of 2007
77 Total content (%) of GM material in each one of the analyzed samples AduanaEstadoRegiónTotal content (%) of GM material in each one of the analyzed samples
78 “Stacked” varieties of Maize Event 1xEvent 2Event 3ACS-ZMØØ3-2MON-ØØ81Ø-6DASNK603TC1507DAS-Ø15Ø7-1MON-ØØ6Ø3-6BT11MIR604GA21LY038MON-ØØ863-5MON-ØØØ21-9MON89034MON88017MON8903SYN-BTØ11-1MON810
79 Evento “Stack” DAS59122-7 x NK603 Copia 1 ADNCopia 2 ADNCopia 3 ADNCopia 4 ADNCopia 5 ADNEvento “Stack” DAS x NK603100%100%200% ?
80 Evento “Stack” DAS59122-7 x NK603 Copia 1 ADNCopia 2 ADNCopia 3 ADNCopia 4 ADNCopia 5 ADNEvento “Stack” DAS x NK60380%100%180% ?
81 Evento “Stack” DAS59122-7 x NK603 Copia 1 ADN(No-GM)Copia 2 ADNCopia 3 ADNCopia 4 ADNCopia 5 ADNEvento “Stack” DAS x NK60380%80%160% ?
83 Use of normalizing gene in tests performed in the laboratory: RT-PCR % OGM = (transgene/normalizing gene) x 100MON810 with hmg15.25 x 100 = 43.834.84MON810 with adh15.25 x 100 = 23.166.01If the amount of endogenous gene increases (denominator), the amount of GMO will be underestimated.
84 Quantification of three events using two different normalizing %Quantification of three events using two different normalizing
85 adh could be present in the genome of the maize in more than one copy. Quantification with adh could underestimate the results
86 Quantification of GM material (total) according to specific events or promoter 35S
87 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 enoughLow astringency PCR programme¿Which endogenous gene should choose?¿Differences between cultivars?
88 ConclusionsExists a high income of GM material in the analyzed customs.The most abundant events were: MON810 > NK603 > DAS1507-1The 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 MexicoThe event MON88017 (authorized variety) was not detected
89 Abraham Acatzi1 Javier Magaña1 Carlos Moles2 Carolina Peña2 Marcela Castillo2Maricarmen Quirasco1Javier Plasencia3Marcelo Signorini4Amanda Gálvez1, 21 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