Application of Sampling and Detection Methods in Agricultural Plant Biotechnology

Description

Application of Sampling and Detection Methods in Agricultural Plant Biotechnology describes detection methods for seed, plants and grain derived from biotechnology. This international handbook, based on a series of workshops carried out for governments in collaboration with ILSI and Co-published in partnership with the Cereals & Grains Association, provides the technical and practical information needed to develop, validate and use detection methods. This useful resource provides readers with the tools necessary to carry out reliable sampling, detection and interpretation of data. Application of Sampling and Detection Methods in Agricultural Plant Biotechnology Copyright Foreword List of contributors 1. Introduction: genetically modified crops and their detection References Further reading 2. Seed purity testing and low-level presence 2.1 Background 2.2 Sampling 2.3 Detection methods and techniques 2.4 Protein-based detection methods 2.5 DNA-based detection methods 2.6 Managing costs in a detection laboratory 2.7 Bioassays 2.8 Low-level presence and purity 2.9 Data analysis and reporting 2.10 Conclusion References 3. Grain supply chain stewardship and testing—import and export 3.1 Introduction 3.2 Testing for import and export 3.2.1 Biotechnology traits in the movement of grain globally 3.2.2 Available methods and suitability for each testing application 3.2.2.1 Protein-based detection methods 3.2.2.2 DNA-based methods 3.2.3 Grain supply chain critical testing points and associated methods 3.2.4 Managing challenges in global grain movement 3.2.5 Emerging challenges and future testing technologies 3.2.5.1 Low-level presence of genetically engineered material 3.3 Regulation and official controls 3.4 Analytical strategy 3.4.1 GMO detection using real-time PCR method 3.4.2 GMO screening 3.4.3 Gene-specific detection 3.4.4 Construct-specific detection 3.4.5 Event-specific detection 3.4.6 Source of reference material 3.5 Stewardship References Further reading 4. Principles of nucleic acid-based detection methods 4.1 Introduction 4.2 DNA preparation and requirements 4.2.1 DNA extraction methods 4.2.2 Requirements of DNA quality 4.3 Nucleic acid hybridization 4.3.1 Southern blot analysis 4.3.2 Probes 4.3.3 Choice of target nucleic acids 4.4 Amplification methods 4.4.1 Polymerase chain reaction 4.4.1.1 Targets for GMO detection 4.4.1.2 Qualitative and quantitative PCRs 4.4.2 Ligase amplification reaction 4.4.3 Transcription-based amplification system 4.4.4 Strand displacement amplification 4.5 Detection platforms 4.6 Next-generation sequencing 4.7 Closing remarks References 5. Method validation: DNA-based detection methods 5.1 Introduction 5.1.1 Method validation standards and guidelines 5.1.1.1 Codex alimentarius guidelines 5.1.1.2 European Union guidelines 5.1.1.3 ISO standards 5.2 PCR validation 5.2.1 Qualitative and quantitative PCR method validation requirements 5.2.1.1 Specificity Definition Acceptance criteria 5.2.1.2 The limit of detection Definition Acceptance criteria 5.2.1.3 Limit of quantification Definition Acceptance criteria 5.2.1.4 Dynamic range Definition Acceptance criteria Quantitative method 5.2.1.5 Trueness (accuracy) Definition Acceptance criteria 5.2.1.6 Precision-relative repeatability standard deviation (RSDr) Definition Acceptance criteria 5.2.1.7 Precision-relative reproducibility standard deviation (RSDR) Definition Acceptance criteria 5.2.1.8 Robustness Definition Acceptance criteria 5.2.1.9 Amplification efficiency Definition Acceptance criteria 5.2.1.10 R2 coefficient Definition Acceptance criteria 5.3 Data interpretation and sources of error 5.3.1 Data interpretation 5.3.2 Source of error 5.3.2.1 General considerations Biological sources of errors Analytical/instrumental sources of errors 5.3.2.2 False positive and false negative False positives False negatives 5.4 Reference materials 5.5 Assay transfer and proficiency testing 5.5.1 Interlab verification 5.5.2 Proficiency testing 5.5.2.1 Technical guidance for PT 5.5.2.2 Programs for PT 5.6 Summary References 6. Protein-based detection methods 6.1 Introduction 6.2 History 6.3 Principle 6.4 Application of immunoassays to GE crops 6.4.1 Why use immunoassays 6.4.2 Immunoassay formats 6.4.3 Sandwich ELISA 6.4.4 Lateral flow strip assay 6.4.5 Other formats 6.5 Production of antibodies 6.5.1 Choice of immunogen 6.5.2 Polyclonal antibodies 6.5.3 Monoclonal antibodies 6.5.4 Recombinant antibodies 6.6 Assay design and operation 6.6.1 Microtiter plate ELISAs 6.6.1.1 ELISA creation and quality control 6.6.1.2 Grinding/tissue disruption 6.6.1.3 Choice of buffer 6.6.1.4 Protein extraction 6.6.1.5 Standard curves 6.6.1.6 Cross-reactivity 6.6.1.7 Determination of the useful range of the assay 6.6.1.8 Other factors 6.6.2 Lateral flow strips 6.6.2.1 LFS creation and quality control 6.6.2.2 LFS for testing single seed or single leaf 6.6.2.3 LFS for testing bulk grain or bulk leaf samples 6.6.2.4 Evolution of the LFS design 6.6.2.5 Validation of a lateral flow assay 6.7 Conclusions Acknowledgments References 7. Protein methods: antibody-based protein method validation and assay verification 7.1 Introduction 7.1.1 Standards and validation parameters 7.2 Quantitative ELISA method validation 7.2.1 Sensitivity 7.2.2 Specificity 7.2.2.1 Cross-reactivity with purified proteins 7.2.2.2 Matrix effects/interferences 7.2.3 Extraction efficiency 7.2.4 Accuracy 7.2.5 Precision 7.2.5.1 Fortified sample approach 7.2.5.2 Dilution linearity 7.3 Independent laboratory validations 7.4 Qualitative method validation 7.4.1 Specificity/selectivity 7.4.1.1 Specificity 7.4.1.2 Selectivity 7.4.2 Sensitivity, establishing the cut-off value (fixed vs. floating) 7.4.2.1 Sensitivity 7.4.2.2 Defining the threshold value 7.4.3 Hook effect 7.4.4 Accuracy 7.4.5 Precision 7.4.6 Ruggedness 7.5 Assay verification 7.5.1 Kit verification 7.5.1.1 Verification of plate-based ELISA kits 7.5.1.2 Verification of lateral flow test strips 7.5.1.3 Critical consumable and reagent verification a) Lot verification of ELISA kits b) Lot verification of lateral flow strips References 8. Reference materials and working standards 8.1 Background 8.2 Uses of reference materials 8.3 What constitutes a reference material? 8.4 Reference materials in agricultural biotechnology 8.5 Practical limitations on reference material 8.6 Sources of reference materials 8.7 Reference material types 8.7.1 Seed 8.7.2 Nonviable seed 8.7.3 Grain/seed powder 8.7.4 Powder produced from nonseed/grain plant parts 8.7.5 Plant DNA 8.7.6 Plasmid DNA 8.7.7 Proteins and plant extracts 8.7.8 Intellectual property issues 8.8 Specific considerations for using reference materials for quantification 8.8.1 True negative or 100% positive reference materials 8.8.2 Zygosity of reference materials 8.8.3 Application of reference materials to stacked events 8.8.4 Uncertainty considerations for quantitative analysis when constructing standard curves 8.9 International coordination and standardization 8.10 Future of reference material and the drivers Acknowledgments References Further reading 9. Seed and grain sampling 9.1 Introduction 9.2 Sampling procedures 9.3 Probabilities of sampling 9.4 Testing of the laboratory sample 9.5 Acceptance sampling with qualitative testing 9.6 Acceptance sampling with quantitative testing 9.7 Acceptance sampling with qualitative testing on multiple subsamples 9.8 Conclusion References 10. Plant and field sampling 10.1 Introduction 10.2 Plant breeding 10.3 Seed and commercial crop testing 10.4 Direct testing in the field 10.5 Sampling for laboratory testing 10.6 Marking and labeling 10.7 Collecting the sample 10.8 Safety considerations 10.9 Specific procedures 10.9.1 Tube sampling 10.9.2 Hand sampling 10.9.3 Disposable test-tube cap method 10.9.4 Core samplers 10.9.5 Tissue punch 10.10 Shipping samples 10.11 Summary 11. Testing laboratory design and management 11.1 Introduction 11.2 Laboratory design 11.3 Laboratory workflow 11.4 Physical laboratory processes 11.5 Equipment management 11.6 Reagents 11.7 Assay quality control 11.8 Personnel management 11.9 Summary References 12. International standards and guidelines 12.1 Background and purpose of standards 12.2 Standards setting process 12.3 Standards organizations 12.3.1 Scientific association SDOs 12.3.2 ISO 12.3.3 Government-led organizations 12.3.3.1 Codex Alimentarius 12.3.3.2 ISTA 12.3.4 National and regional organizations 12.3.4.1 National organizations 12.3.4.2 Regional organizations 12.4 Development of a standard 12.5 Standards relevant to GM detection 12.5.1 Sampling 12.5.2 Guidelines and standards regarding analytical methods 12.5.3 General analytical methods standards 12.5.4 Sources of methods 12.5.5 Laboratory accreditation standards 12.6 Conclusions Acknowledgment References Further reading 13. Analytical strategy and interpretation of results 13.1 Introduction 13.2 Confirming the presence of intended trait 13.2.1 Herbicide bioassay 13.2.1.1 Introduction 13.2.1.2 Methodologies 13.2.1.3 Seedling morphology of susceptible seedlings 13.2.1.4 Training resources 13.2.2 Immunoassay 13.2.2.1 Enzyme-linked immunosorbent assay 13.2.2.2 Lateral flow strips 13.2.2.3 Other immunoassay formats Meso scale discovery Microbead-based array-Luminex 13.3 Confirming the absence of unintended traits 13.3.1 Immunoassay 13.3.1.1 Enzyme-linked immunosorbent assay 13.3.1.2 Lateral flow strips 13.3.1.3 Other formats Antibody-based homogeneous proximity assays Spatial proximity analyte reagent capture luminescence Antibody-free detection methods 13.4 Interpreting results 13.4.1 Introduction 13.4.2 Qualitative test 13.4.3 Quantitative test References 14. Detection methods for genome-edited crops 14.1 Introduction 14.2 What is genome editing—the key technologies 14.3 The drivers for detection methods 14.4 Detection method approaches 14.4.1 PCR 14.4.1.1 Challenges 14.4.2 Digital PCR 14.4.2.1 Challenges 14.4.3 Sequencing 14.4.3.1 Challenges 14.5 Differentiation of genome edits and spontaneous or induced untargeted mutations 14.6 Conclusions References 15. Future perspectives and challenges 15.1 Introduction 15.1.1 Issues and challenges 15.2 New GMO detection technologies 15.2.1 DNA-based technologies 15.2.1.1 qPCR-based multiplex strategies 15.2.1.2 Digital PCR technology 15.2.1.3 Isothermal amplification 15.2.1.4 Next-generation sequence 15.2.2 Protein-based technologies 15.2.2.1 LC-MS multiplexing technology 15.2.2.2 Immunoassays MSD multiplexing array (electrochemical luminescence) Biacore biosensor system (piezo-electric) High throughput AlphaLISA (optical through FRET) Bead-based multiplex technology (optical—fluorescence with flow cytometry) 15.3 Closing remarks References Further reading Index A B C D E F G H I J K L M N O P Q R S T U V W Z