The introduced rdna sequence differentiates the GM plant from its non-gm counterpart

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3 The introduced rdna sequence differentiates the GM plant from its non-gm counterpart J. Zel et al., How to reliably test for GMOs, SpringerBriefs in Food, Health, and Nutrition DOI /

4 Detection of GMOs is required for: Biotech producers, seed companies, farmers To assist variety conversion To track different products To assure compliance of products to commercial purity standards Seed companies, farmers, food & feed industry, enforcement authorities, testing laboratories To monitor products after commercial release To assure segregation of products To check compliance with legislation (regulated vs unregulated, labeling thresholds)

5 GMO analysis. Does the product contain GMO or not? A positive/negative statement. Which GMO is present? Is it regulated or deregulated? Determine the GMO content (%) Compliance to threshold regulation.

6 . Sampling GMO detection Authorised? GMO identification Positive Negative Yes No Illegal identify unauthorised No action required GMO Quantification <Threshold >Threshold No labelling Labelling required

7 Detection of GMOs GMOs can be identified by detecting the inserted genetic material at several levels:

8 Evolution of GMO Analysis and associated reference materials Holst-Jensen, Biotechnology Advances 27 (2009)

9 Methods of GMO detection 1. Bioassays 2. Protein-based methods ELISA Lateral flow device DNA-based methods End-point PCR Real-time PCR Microarrays Other

10 Protein based GMO detection This immunoassay method (ELISA) gives a present /absent result Proteins EPSPS, CRY VIP3A GMO - Pr otein Antibody Antibody Label Easy to perform, cheap and reliable Test results within few minutes Best for raw agricultural commodities (leaf, seeds/grains) Identification of a specific event not possible It is possible to obtain a 99% confidence level of less than 0.15% GMO for a given lot.

11 Lateral Flow Strip Test : Herbicide Tolerance O.2g sample ml Tap H 2 O Shake vigorously until entire sample is wet (20-30s) Control Line Test Line Let stand (30s) Insert Lateral Flow test strip 0% 0.01% 0.1% 0.5% 1% Read after 5 mins

12 DNA-based testing Non-GMO GMO

13 DNA Based Methods: PCR 1. DNA Extraction 2. Amplification 3. Electrophoresis

14 Detection of GMOs: Presence or Absence Screening methods target a part of rdna sequence that is present in many GMOs, such as the regulatory sequences of promoters, terminators and construct specific. J. Zel et al., How to reliably test for GMOs, SpringerBriefs in Food, Health, and Nutrition DOI /

15 Real Time PCR for Screening GMOs:p35S Positive sample Negative sample Ct value

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17 Identification of GMOs Performed with event-specific methods usually targeting the nucleotide sequence at the junction between the plant host genome and the rdna unique to an individual GMO, allowing identification of the GMO present in the sample J. Zel et al., How to reliably test for GMOs, SpringerBriefs in Food, Health, and Nutrition DOI /

18 Quantification of identified GMO Quantification of GMO is done by measuring the ratio between taxonspecific sequence (House keeping gene) and event-specific sequence

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20 ?Ct Amplification of EPSPS gene Amplification of lectin gene GM Soy Analysis Std Curve y = Ln(x) R = % GM Calculated Std Curve Quantitative detection of Roundup Ready Soybean

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22 Complexity of GMO Analysis Types of Analysis Types of samples

23 Harmonisation of GMO Analysis X % Y % X % Z % Z %

24 Harmonisation of GMO Analysis X % X % X % X % X %

25 GMO Testing : Unidirectional route of sample One of the most important precautions in GMO testing is to prevent cross contamination. Separate rooms (or chambers) for each operation 1. Sample receipt, 2. Homogenization, 3. DNA extraction PCR ± 3 mix C) preparation, 5. Addition of the extracted DNA coats to the PCR reaction wells, Room temperature control (e.g., Changing gloves and laboratory 6. Using Real disposable time PCR plasticware Separate reaction reagents and pipette sets, for each room

26 Accreditation to ISO 17025: 2017 Accreditation is based on criteria and procedures specially conceived for technical competency evaluation Accreditation will verify the similarity to standard requirements and will also prove the technical competency of a testing laboratory It is a validation of competency and relevance of a quality organisation. Being accredited is a proof of trust for regulatory bodies, companies, clients, consumers It is a precious tool to facilitate and accelerate international exchanges (export/import)

27 Technical requirements for detection of GMOs: Standards

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29 Reliability and Quality of Analytical Output Adapted from J. Zel et al., How to reliably test for GMOs, SpringerBriefs in Food, Health, and Nutrition DOI /

30 Verification of the method 1. Accuracy 2. Specificity 3. Applicability (Matrix And Concentration Range) 4. Limit Of Detection; 5. Limit Of Determination; 6. Precision 7. Robustness 1. Repeatability; 2. Reproducibility 3. Recovery 4. Selectivity 5. Sensitivity 6. Linearity 7. Measurement Uncertainty

31 Quality control in GMO testing

32 Certified Reference Materials for GMO analysis Reference materials are used as positive controls for GMO analysis Accompanied with certificate on its quality and origin. GMO content is given in mass fraction and, where available, in copy number per haploid genome equivalent

33 Flow Chart for GMO Analysis NON GMO (CRM) (-ve Control) GMO STANDARD (CRM) (+ve Control) Homogenisation & DNA Extraction TEST MATERIAL CTAB Method DNA Quality Column/Kits Quantify DNA End point PCR Real Time PCR Interpretation & Documentation

34 Sample homogenisation Homogenization is required for two reasons: 1. To achieve sufficient efficiency of DNA extraction from different matrices 2. To ensure homogeneity and equal representation of GMO-derived particles in the subsamples Achieved with mills, homogenizers, immersion blenders, coffee grinders, or a suitable equivalent device, depending on the size and the structure of the laboratory sample SOP for grinding

35 Acceptance criteria for extracted DNA 1. DNA concentration: should be appropriate for the subsequent PCR analyses. Example: if the PCR protocol indicates 40 ng/µl as the concentration of the DNA solution to be added to the master-mix, the average concentration of the DNA extract should be > 40 ng/µl) 2. Yield: should be at least as much as is required for the subsequent PCR analyses. The DNA extraction module should provide similar yields for both GM and non-gm material on the same matrix. 3. The minimum size of the majority of DNA fragments should be larger than the size of the amplicon produced by the PCR module used in subsequent analyses.

36 DNA Yield: Processing method Vijaykumar et al, Food Chemistry (2009) 117,

37 PCR Efficiency: DNA extraction Cankar, et al., BMC Biotechnology 2006, 6:37 doi: /

38 PCR efficiency: Relative absence of PCR inhibitors Realative absence of PCR inhibitors Three conditions should be met: 1. The slope of the regression line must be between -3.6 and -3.1; 2. The coefficient of determination (R 2 is equal to or above 0.98; 3. The difference between measured Ct and extrapolated Ct value (ΔCt) is below 0.5 for the undiluted sample.

39 An ideal qpcr for GMO analysis A ideal Real-time PCR experiment will have the following characteristics: Dilution series has expected spacing Curves are all S- shaped Plateau height doesn t matter Replicates are tightly clustered Curves are smooth Baselines are relatively flat Melt curve has one peak per product.

40 Use of control charts J. Zel et al., How to reliably test for GMOs, SpringerBriefs in Food, Health, and Nutrition DOI /

41 Reliable results J. Zel et al., How to reliably test for GMOs, SpringerBriefs in Food, Health, and Nutrition DOI /

42 One sees what one wants to see..