Testing and analysis of GMO-containing foods and feed

Cover
Half Title
Title Page
Copyright Page
Dedication
Contents
Preface
Editors
List of Contributors
SECTION 1 GMOs
	Chapter 1 Genetically Modified Organisms
		1.1 What are Genetically Modified Organisms?
		1.2 GMOs and Biotechnology
		1.3 How are GMOs Produced?
		1.4 The Controversy about GMOs
			1.4.1 Proponents and Opponents of the GM Technology
			1.4.2 Arguments in Favor of GMOs and GM Foods
			1.4.3 Arguments against GMOs and GM Foods
		1.5 Uses of GMOs
			1.5.1 Uses of GMOs in Agriculture
			1.5.2 Uses of GMOs in Medicine and the Pharmaceutical Industry
			1.5.3 Uses of GMOs in Bioremediation
		1.6 Regulation of GMOs
			1.6.1 Regulation-General Aspects
			1.6.2 Substantial Equivalence (SE)
			1.6.3 Labeling
		1.7 Public Awareness on GMOs
			1.7.1 Public Awareness on GMO Products, with Special Reference to GM Foods
			1.7.2 Roles of Mass Media
		1.8 Recent Developments on GMOs
		References
		Suggested References
SECTION 2 SAMPLING
	Chapter 2 Guidelines for Sample Preparation Procedures in GMO Analysis
		2.1 Introduction
		2.2 Scope
		2.3 Principle
		2.4 Definitions
			2.4.1 General Definitions
				2.4.1.1 Lot
				2.4.1.2 Grab Sampling
			2.4.2 Definitions Concerning the “Sample”
				2.4.2.1 Laboratory Sample
				2.4.2.2 Reserve Sample
				2.4.2.3 Test Portion
				2.4.2.4 Test Sample
				2.4.2.5 Other Definitions Related to the Sample
			2.4.3 Definitions Concerning the “Sample Preparation Procedure”
				2.4.3.1 Coarse Grinding (or Pre-grinding)
				2.4.3.2 Comminution
				2.4.3.3 Constitutional (Compositional) Heterogeneity
				2.4.3.4 Distributional Heterogeneity
				2.4.3.5 Fragment—Particle
				2.4.3.6 Homogeneity
				2.4.3.7 Increment
				2.4.3.8 Incrementing (Composite Sampling)
				2.4.3.9 Mass Reduction
				2.4.3.10 Mixing–blending-Homogenization
				2.4.3.11 Particle Size Reduction
		2.5 General Considerations Related to the Theory of Sampling
			2.5.1 Errors Linked to Characteristics of the Material under Analysis
				2.5.1.1 Constitutional Heterogeneity
				2.5.1.2 Distributional Heterogeneity
			2.5.2 Errors linked to the Subsampling Process
				2.5.2.1 Increment Delineation Errors
				2.5.2.2 Increment Extraction Errors
				2.5.2.3 Increment Preparation Errors
			2.5.3 Subsampling Operations
		2.6 Equipment
			2.6.1 Cleaning Tools
			2.6.2 Sampling Tools
			2.6.3 Sample Preparation
			2.6.4 Drying Systems
			2.6.5 Equipment for Particle Size Reduction and Mixing
			2.6.6 Equipment for the Storage of Samples
		2.7 Environment and Rooms
			2.7.1 Rooms
			2.7.2 Cleaning and Decontaminating Reagents
		2.8 Safety Precautions
		2.9 Procedure
			2.9.1 Laboratory Sample Check
				2.9.1.1 Check of Laboratory Sample Constitution
				2.9.1.2 Maximum Mass of the Laboratory Sample
			2.9.2 Mass Reduction
				2.9.2.1 Coarse Grinding (or Pre-grinding)
				2.9.2.2 Subsampling Procedures
			2.9.3 Particle Size Reduction
				2.9.3.1 General Methods
				2.9.3.2 Requirements Regarding the Choice of Size Reduction Equipment
				2.9.3.3 Maintaining Integrity of the Laboratory Sample
				2.9.3.4 Mixing Techniques
			2.9.4 Partial Drying
			2.9.5 Special Sample Preparation Procedures
				2.9.5.1 Gelatine, Molasses, and Samples with High Fat Content
				2.9.5.2 Viscous Samples
				2.9.5.3 Liquid Samples
				2.9.5.4 Plant Tissues
			2.9.6 Test Portion
				2.9.6.1 Homogenization of the Test Sample (or Analytical Sample) Prior to Test Portion Uptake
				2.9.6.2 Minimum Mass of Test Portion
				2.9.6.3 Test Portion Uptake
			2.9.7 Storage and Disposal
		2.10 Performance Tests (Quality Control)
			2.10.1 Performance Test for Particle Size Reduction (Grinding)
				2.10.1.1 Grinding Quality
				2.10.1.2 Carry-over
			2.10.2 Performance Test for Mixing
				2.10.2.1 Unmixed Layers Method
			2.10.3 Environmental Control
			2.10.4 Representativeness of the Test Portion
			2.10.5 Overall Performance Test on Sample Preparation Steps through a Replication Experiment
		2.11 Identified Gaps and Conclusions
		Bibliographic References
		Appendix A: Definitions Related to the Concept of “Sample” According to Standards and European Legislation
		Appendix B: Examples of Grinders Used by Laboratories Involved in GMO Detection
		Appendix C: Flow Chart of the Successive Steps of the Sample Preparation Procedure for Particulate Material
		Appendix D: Mathematical Formula that Might Be Very Helpful in GMO Detection for Determination of the Laboratory Sample Size When Testing Kernels
		Appendix E: List of Food Matrices that Do Not Need Homogenization or Grinding
		Appendix F: The Paper-Cone Riffle Splitter (for Information Only)
		Appendix G: Replication Experiment
SECTION 3 GMO TESTING METHODS
	Chapter 3 Immunoassays (Protein-Based Methods)
		3.1 Key Components of Immunoassay
		3.2 Enzyme-linked Immunosorbent Assay
		3.3 ELISA Tests for GM Crops
		3.4 Dipstick or Immunochromatographic Strip
			3.4.1 Components of Dipstick
				3.4.1.1 Sample Pad
				3.4.1.2 Conjugate Pad (Release Pad)
				3.4.1.3 Membrane
				3.4.1.4 Absorbent Pad (Sink Pad)
				3.4.1.5 Labels (Probes)
			3.4.2 Principle of Dipstick Test
			3.4.3 Dipsticks for Detection of GM Content
			3.4.4 Multiplex Dipsticks for Simultaneous Detection of Transgenic Proteins
			3.4.5 Advances in Development of Dipsticks
			3.4.6 Factors Affecting Dipstick Assay
		3.5 Immuno-PCR Method
		3.6 Advantages and Disadvantages of Immunoassays
		3.7 Concluding Remarks
		References
	Chapter 4 PCR Techniques for Detection and Quantification of GMOs
		4.1 Introduction
			4.1.1 Classical PCR
			4.1.2 Nested PCR
			4.1.3 Real-Time PCR
				4.1.3.1 The Cq concept
			4.1.4 Multiplex PCR
			4.1.5 Digital PCR
		4.2 GMO Analysis and Types of Targets Amplified
			4.2.1 Universal Plant Targets
			4.2.2 Targets Specific to Plant Species
				4.2.2.1 Sugar Beet (Beta vulgaris)
				4.2.2.2 Wheat (Triticum aestivum)
				4.2.2.3 Chicory (Cichorium intybus)
				4.2.2.4 Rapeseed (Brassica napus)
				4.2.2.5 Cotton (Gossypium hirsutum)
				4.2.2.6 Bean (Phaseolus vulgaris)
				4.2.2.7 Flax (Linum usitatissimum)
				4.2.2.8 Maize (Zea mays)
				4.2.2.9 Papaya (Carica papaya)
				4.2.2.10 Pea (Pisum sativum)
				4.2.2.11 Potato (Solanum tuberosum)
				4.2.2.12 Rice (Oryza sativa)
				4.2.2.13 Soybean (Glycine max)
				4.2.2.14 Tomato (Solanum lycopersicum)
				4.2.2.15 Sunflower (Helianthus annuus)
			4.2.3 Screening Targets
				4.2.3.1 Screening Focused on Promoters and Terminators
				4.2.3.2 Screening Focused on Gene Coding Sequences
				4.2.3.3 Screening and Donor Organisms
				4.2.3.4 Event-Specific Targets
		4.3 GMO Quantification
			4.3.1 Quantification by qPCR
				4.3.1.1 Delta Cq Method
				4.3.1.2 Dilutions of Calibrants
			4.3.2 Quantification by Digital PCR
			4.3.3 Considerations on the Targets Used for Quantification
			4.3.4 Considerations on the Unit of Measurement
		4.4 Conclusion
		References
	Chapter 5 Sensors
		5.1 Biosensors
		5.2 Classification of Biosensors
		5.3 Recognition Event
			5.3.1 Catalytic Biosensors
			5.3.2 Affinity Biosensors
				5.3.2.1 Immunosensors
				5.3.2.2 Nucleic Acid-Sensors
				5.3.2.3 Aptasensors
		5.4 Methods of Immobilization
		5.5 Transduction Principle
			5.5.1 Electrochemical Biosensors
				5.5.1.1 Voltammetric
				5.5.1.2 Potentiometric
				5.5.1.3 Conductimetric/Impedimetric
				5.5.1.4 Chemical-sensitive Field Effect Transistor
			5.5.2 Optical Biosensors
				5.5.2.1 Absorbance
				5.5.2.2 Fluorescence
				5.5.2.3 Chemo/Electro/Bioluminescence
				5.5.2.4 Evanescent Wave Biosensors
			5.5.3 Mass-sensitive Biosensors
			5.5.4 Thermometric Biosensors
			5.5.5 Nanomechanic Biosensors
		5.6 Biosensors as Platforms for Genetically Modified Organism Detection
			5.6.1 Electrochemical Biosensors
				5.6.1.1 DNA Biosensors
				5.6.1.2 Immunosensors
			5.6.2 Optical Biosensors
				5.6.2.1 SPR Biosensors
				5.6.2.2 SERS Biosensors
				5.6.2.3 Chemiluminescence Biosensors
				5.6.2.4 Visual Biosensors
			5.6.3 Piezoelectric Biosensors
		5.7 Final Remarks
		References
	Chapter 6 The Application of Nucleic Acid Microarrays for the Detection of Genetically Modified Organisms
		6.1 Introduction
		6.2 Nucleic Acid Microarray Technology
			6.2.1 Essential Features
			6.2.2 A Brief History of GMO Microarrays
			6.2.3 Hybridization Biosensors
		6.3 Technical Considerations for a Successful Microarray Approach
			6.3.1 Sample Preparation and Microarray Design
			6.3.2 Target DNA Amplification
			6.3.3 Labeling and Detection
			6.3.4 Remaining Technical Challenges
		6.4 Future Prospects for Microarray-based GMO Detection
			6.4.1 Detection of Unknown GMOs and Edited Genomes
			6.4.2 Where Next for GMO Microarray Analysis?
		References
	Chapter 7 Recent Developments in Detection Methods of Genetically Modified Organisms
		7.1 Introduction
		7.2 Real-time Polymerase Chain Reaction Multiplex Strategies
		7.3 Pre-spotted Real-time PCR Plates
		7.4 PCR Capillary Gel Electrophoresis (PCR-CGE)
		7.5 Digital PCR
		7.6 Loop-mediated Isothermal Amplification
		7.7 Luminex Technology
		7.8 Microarray-based technology
		7.9 DNA Sequencing-based approaches
			7.9.1 Pyrosequencing
			7.9.2 DNA Walking
			7.9.3 Next-Generation Sequencing
			7.9.4 Whole Genome Sequencing
		7.10 Sensors
		7.11 Other Detection Methods
			7.11.1 Chromatography and Mass Spectrometry
			7.11.2 Near Infrared Spectroscopy
			7.11.3 Terahertz Spectroscopy
		References
SECTION 4 VALIDATION AND STANDARDIZATION OF METHODS
	Chapter 8 Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods
		8.1 Introduction
		8.2 Terminology
			8.2.1 Validation of Method
			8.2.2 Verification of Method
			8.2.3 Precision—Relative Repeatability Standard Deviation (RSDr)
			8.2.4 Laboratory Sample (see also Fig. 1)
			8.2.5 Analytical Sample
			8.2.6 Test Portion
			8.2.7 Test Result
			8.2.8 DNA Extraction Replicates (as Used in this Chapter)
			8.2.9 PCR Replicates (as Used in this Chapter)
			8.2.10 Working Dilution
			8.2.11 Limit of Quantification (LOQ)
			8.2.12 Practical Limit of Quantification (Practical LOQ)
			8.2.13 Limit of Detection
			8.2.14 Practical Limit of Detection (Practical LOD)
			8.2.15 Specificity
			8.2.16 Dynamic Range
			8.2.17 Trueness
			8.2.18 Amplification Efficiency
			8.2.19 R[sup(2)] Coefficient
			8.2.20 Robustness
		8.3 General Considerations
		8.4 DNA Extraction and Purification
			8.4.1 DNA Concentration
			8.4.2 Absence of Inhibitors in the DNA Extracts
		8.5 Specificity
		8.6 Dynamic Range, R[sup(2)] Coefficient, and Amplification Efficiency
		8.7 Trueness
		8.8 RSDr
		8.9 Estimation of the LOQ
		8.10 Estimation of Limit of Detection (LOD)
		8.11 Robustness
		8.12 Relevant Samples
		Appendix A: Effect of DNA Content on the Practical LOD
		Appendix B: Evaluation of DNA—Extraction Method (Inhibition Test)
		Appendix C: Production of Intermediate Concentrations of Positive Material
		Appendix D: Estimation of the Mean, Standard Deviation and Relative Repeatability Standard Deviation of GM-Content from Real-Time PCR
		References
	Chapter 9 Reference Materials for GMO Analysis
		9.1 GMO Reference Materials
		9.2 Available GMO Reference Materials
		9.3 Development of GMO Reference Materials
		9.4 Use of GMO Reference Materials
		9.5 Who Does What on GMOs in the JRC?
		9.6 Request for the Development of a Set of GMO CRMs
		9.7 American Oil Chemists’ Society and Fluka/Sigma-Aldrich
		9.8 U.S. Food and Drug Administration
		References
	Chapter 10 ISO (International Organization for Standardization)
		References
	Chapter 11 Reference Methods for GMO Analysis
		11.1 EU Database of Reference Methods for GMO Analysis
		11.2 Submission of Method for Validation to ENGL Advisory Group
		11.3 GMO Detection Method Database
		11.4 GMOfinder
		11.5 JRC GMO-Matrix Application
		11.6 The CropLife International Detection Methods Database
		References
Index