DNA Techniques to Verify Food Authenticity : Applications in Food Fraud. 9781788016025, 1788016025

Table of contents :
Cover
DNA Techniques to Verify Food Authenticity: Applications in Food Fraud
Foreword
Preface
Introduction
Contents
Chapter 1 --
The Role of DNA Analysis in the Determination of Food Authenticity
1.1 A Brief History of Food Adulteration
1.2 Food Fraud in the 21st Century
1.3 Challenges in Detecting Food Misdescription and Fraud
1.4 The Role of DNA in Food Authenticity Determination
1.5 Application of DNA-based Analytical Methods to Different Commodities and Food Authenticity Problems 1.6 Pushing the Boundaries: Specialist Techniques for Breed or Variety Identification and Determination of Geographical Origin1.7 Fitness for Purpose
1.8 Final Comments
References
Chapter 2 --
Forensic DNA --
Criminal and Paternity Methods and Applications --
How Can This Help in Verifying Food Authenticity†
2.1 Some History
2.2 Applying the Forensic Code of Conduct
2.3 Key Issues --
Complexities Within Forensic Regulatory Bodies and Associations
2.4 Key Issues --
Databases and Communication
2.5 Document Recording
2.5.1 Receipt of Materials 2.5.2 Examination, Standard Operating Procedures and Quality Control2.6 Conclusion
References
Chapter 3 --
DNA Extraction from Food Matrices†
3.1 Introduction
3.2 Factors Influencing the Choice of Extraction Methodology
3.2.1 Sample Source and Processing
3.2.2 Sample Collection and Storage
3.2.3 Homogenisation
3.2.4 Presence of Contaminants
3.2.4.1 Proteins and RNA
3.2.4.2 Polysaccharides
3.2.4.3 Polyphenols
3.3 General DNA Extraction Methods
3.3.1 Phenol-Chloroform Extractions
3.3.2 Detergent and Protease-based Extractions
3.3.3 Solid-phase Extraction Methods 3.3.4 Concentrating DNA Post-extraction3.3.4.1 Concentrating Using Ethanol Precipitation
3.3.4.2 Concentrating Using a Microfiltration Device
3.4 DNA Extraction Methods Frequently Employed with Food and Feed Samples
3.5 Determining DNA Quantity and Purity
3.5.1 Quantification of DNA
3.5.2 Evaluation of DNA Purity
3.5.2.1 A260:A280 Ratio to Evaluate Levels of Protein Contamination
3.5.2.2 A260:A230 Ratio to Evaluate Levels of Contamination
3.5.3 RNA Contamination
3.5.4 Non-nucleic- acid Contamination
3.5.5 Evaluation of a DNA Sample's Integrity 3.5.6 Determining the Presence of Inhibitory Compounds3.6 Concluding Remarks
Acknowledgements
References
Chapter 4 --
"DNA Techniques" Case Study: Isothermal Approaches
4.1 Introduction
4.2 Main Isothermal NAA Technologies
4.2.1 Cross Priming Amplification (CPA)
4.2.1.1 Overview
4.2.1.2 Benefits
4.2.1.3 Technology Considerations
4.2.2 Helicase-dependent Amplification (HDA)
4.2.2.1 Overview
4.2.2.2 Benefits
4.2.2.3 Technology Considerations
4.2.3 Loop-mediated Isothermal Amplification (LAMP)
4.2.3.1 Overview
4.2.3.2 Benefits
4.2.3.3 Technology Considerations

Citation preview

DNA Techniques to Verify Food Authenticity Applications in Food Fraud

Food Chemistry, Function and Analysis Series editors:

Gary Williamson, University of Leeds, UK Alejandro G. Marangoni, University of Guelph, Canada Juliet A. Gerrard, University of Auckland, New Zealand

Titles in the series:

1: Food Biosensors 2: Sensing Techniques for Food Safety and Quality Control 3: Edible Oil Structuring: Concepts, Methods and Applications 4: Food Irradiation Technologies: Concepts, Applications and Outcomes 5: Non-­extractable Polyphenols and Carotenoids: Importance in Human Nutrition and Health 6: Cereal Grain-­based Functional Foods: Carbohydrate and Phytochemical Components 7: Steviol Glycosides: Cultivation, Processing, Analysis and Applications in Food 8: Legumes: Nutritional Quality, Processing and Potential Health Benefits 9: Tomato Chemistry, Industrial Processing and Product Development 10: Food Contact Materials Analysis: Mass Spectrometry Techniques 11: Vitamin E: Chemistry and Nutritional Benefits 12: Anthocyanins from Natural Sources: Exploiting Targeted Delivery for Improved Health 13: Carotenoid Esters in Foods: Physical, Chemical and Biological Properties 14: Eggs as Functional Foods and Nutraceuticals for Human Health 15: Rapid Antibody-­based Technologies in Food Analysis 16: DNA Techniques to Verify Food Authenticity: Applications in Food Fraud

How to obtain future titles on publication:

A standing order plan is available for this series. A standing order will bring delivery of each new volume immediately on publication.

For further information please contact:

Book Sales Department, Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge, CB4 0WF, UK Telephone: +44 (0)1223 420066, Fax: +44 (0)1223 420247 Email: [email protected] Visit our website at www.rsc.org/books

DNA Techniques to Verify Food Authenticity Applications in Food Fraud Edited by

Malcolm Burns

LGC Ltd, UK Email: [email protected]

Lucy Foster

Department for the Environment Food and Rural Affairs, UK Email: [email protected] and

Michael Walker

Michael Walker Consulting Ltd, UK Email: [email protected]

Food Chemistry, Function and Analysis No.16 Print ISBN: 978-­1-­78801-­178-­5 PDF ISBN: 978-­1-­78801-­602-­5 EPUB ISBN: 978-­1-­78801-­897-­5 Print ISSN: 2398-­0656 Electronic ISSN: 2398-­0664 A catalogue record for this book is available from the British Library © The Royal Society of Chemistry 2020 All rights reserved Apart from fair dealing for the purposes of research for non-­commercial purposes or for private study, criticism or review, as permitted under the Copyright, Designs and Patents Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry or the copyright owner, or in the case of reproduction in accordance with the terms of licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page. Whilst this material has been produced with all due care, The Royal Society of Chemistry cannot be held responsible or liable for its accuracy and completeness, nor for any consequences arising from any errors or the use of the information contained in this publication. The publication of advertisements does not constitute any endorsement by The Royal Society of Chemistry or Authors of any products advertised. The views and opinions advanced by contributors do not necessarily reflect those of The Royal Society of Chemistry which shall not be liable for any resulting loss or damage arising as a result of reliance upon this material. The Royal Society of Chemistry is a charity, registered in England and Wales, Number 207890, and a company incorporated in England by Royal Charter (Registered No. RC000524), registered office: Burlington House, Piccadilly, London W1J 0BA, UK, Telephone: +44 (0) 20 7437 8656. Visit our website at www.rsc.org/books Printed in the United Kingdom by CPI Group (UK) Ltd, Croydon, CR0 4YY, UK

Foreword The European horse meat scandal of 2013 refocused global attention on food authenticity. Food fraud inflicts harm on consumers and businesses, eroding confidence in the integrity of our global food system. Reputations can be badly damaged, food safety compromised and money and trust lost. The subsequent review I led introduced the term ‘food crime’ into the lexicon and my recommendations were accepted in full by the UK Government. These included the development of methods, their validation and sustainable laboratory services as key components. This book is edited by a distinguished trio of scientists who each played a significant role in dealing with the horse meat scandal and its aftermath. They have recruited the top experts in the fields of molecular biology and food authenticity across the globe to contribute what will, I am sure, become the definitive text on DNA techniques to verify food authenticity. They have fulfilled their aims to provide a fundamental understanding of modern DNA techniques applied to food and to reflect current issues and cutting-­edge techniques, such as next generation sequencing and digital PCR. They enable the reader to choose optimum techniques and approaches to address various aspects of food authenticity amenable to DNA techniques. They have also considered wider aspects around harmonisation, standardisation and collaboration, which are intrinsic to ‘fit for purpose’ analysis to protect consumers, enforce food labelling law and maintain a competitive and resilient food sector. The book will serve as an informative reference text for new users and provide a touchstone for best practice for experienced scientists. I congratulate

  Food Chemistry, Function and Analysis No.16 DNA Techniques to Verify Food Authenticity: Applications in Food Fraud Edited by Malcolm Burns, Lucy Foster and Michael Walker © The Royal Society of Chemistry 2020 Published by the Royal Society of Chemistry, www.rsc.org

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the editors and contributors for successfully compiling this excellent volume that describes the state of the art of DNA-­based approaches for the detection and prevention of food fraud and food crime. Christopher Elliott OBE Founder of the Institute for Global Food Security Queen's University of Belfast

Preface When asked about writing or editing a book those who have done so generally reply “Don't do it”. This has not been our experience. When Nicki Dennis first approached one of us (Michael Walker) on behalf of the Royal Society of Chemistry to put together a book on DNA and food authenticity the reaction was – what a great idea, but I can't do this on my own. Two experts, Lucy Foster and Malcolm Burns, immediately sprang to mind and both readily agreed to participate. What followed was two years of hard work. But we can honestly say the experience has been one of exciting learning – from each other and from the subject matter experts who have generously contributed chapters. As a team of editors we appear to have complementary skills and networks so that the whole process has been one of collaborative enjoyment. The Royal Society of Chemistry team, Katie Morrey, Janet Freshwater and Robin Driscoll have been helpful and cooperative throughout. A particular mention must go to Dr Sandy Primrose. Sandy's vision is to bring the quality assurance rigour now commonplace in analytical chemistry to the younger discipline of molecular biology. This vision forms a core theme of the book and Sandy provided a guiding hand throughout, as well as contributing several chapters as a long-­standing authority on food authenticity. In terms of the expertise that we bring to this book, we all have food analysis in common. Dr Malcolm Burns trained as a plant molecular biologist at Birmingham University, gaining his PhD in quantitative trait loci mapping in Arabidopsis in 1997. Following a move to LGC Ltd after graduating, he has built his career in molecular biology, acting as the Principal Scientist for food authenticity testing, specialising in using and advising on advanced DNA approaches for food analysis at a national and international level.

  Food Chemistry, Function and Analysis No.16 DNA Techniques to Verify Food Authenticity: Applications in Food Fraud Edited by Malcolm Burns, Lucy Foster and Michael Walker © The Royal Society of Chemistry 2020 Published by the Royal Society of Chemistry, www.rsc.org

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Dr Lucy Foster [Institute of Food Safety and Technology (IFST) Fellow] trained as a food scientist, gaining her PhD in analytical and nutritional aspects of selenium deficiency in 1997. Following research roles at Unilever (Colworth) and the Institute of Food Research (Norwich) she joined the Ministry of Agriculture Fisheries and Food to work in food authenticity,  and subsequently the Food Standards Agency to lead work in a variety of roles, including food additives and foodborne disease policy and running food science research and surveillance programmes. Following a move to the Department for the Environment, Food and Rural Affairs (Defra) in 2009 to lead agriculture and food chain research, she currently heads Defra's Agri-­Food Chain Directorate's teams on food science, innovation, GM and genetic resources policy. Dr Michael Walker is a Chartered Chemist and Fellow of both the IFST and the Royal Society of Chemistry, and holds the MChemA, the statutory qualification to act as a Public Analyst in the UK. He owns a thriving chemico-­legal private practice, and is also Referee Analyst and Head of the Office of the Government Chemist in the UK National Measurement Laboratory hosted in LGC, a member of the European Academy of Allergy & Clinical Immunology, and a member of the IFST Scientific Committee. He was a subject matter expert to the UK ‘Elliott Review’ in the aftermath of the horse meat scandal and was a founder board member of the UK Food Standards Agency. We have worked together over many years on food authenticity issues, but it was the horse meat incident in 2013 which threw us together in a unique way to deal with a variety of analytical challenges to meet the needs of government, industry and food law enforcers and consumers. This resulted in new ways of thinking about metrology, quantitative DNA analysis for food analysis, how this affects food law enforcement and gaps in understanding around some of the challenges using DNA applications. These gaps  included ensuring fitness for purpose, harmonisation and standardisation of DNA methods. The incident also highlighted the need for greater awareness-­ raising of the existing and emerging analytical tools that are available to the wider community, as well as the potential for genomic approaches in the future as a tool for detecting food fraud. Discussions during a Government Chemist conference dinner in 2016 crystallised the concept for a book to address these issues. This book provides, we trust, a valuable resource for learning more about the scope, application, benefits and pitfalls associated with DNA-­based techniques in one comprehensive volume. It is unique in that it gathers a wide array of internationally recognised scientists, all experts in their fields of study, to provide authorship on topical and cutting-­edge perspectives for food authenticity testing as well as providing insight into requirements for the future. The book is aimed at those who wish to understand the fundamental principles of the application of the DNA-­based approaches in the laboratory environment, those who are already practicing the techniques but wish to further refine their skill sets, and those who wish to learn more about the

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new and emerging techniques for food authenticity testing based on genomic approaches on the horizon. As such, we hope that the book will appeal to the food industry, official control laboratories, researchers, and students and experienced practitioners alike. As editors we are indebted to all the chapter authors who have given up their valuable time and expertise to share their knowledge. They have worked with us enthusiastically to provide unparalleled breadth and depth of discussion. We would also like to thank colleagues in the Molecular Biology team at  LGC and Defra's Food Science team, and Professor Sir Ian Boyd (Chief  Scientific Advisor, Defra) for their contributions, support and encouragement One of us (MB) also gratefully acknowledges funding from the UK Department for Business, Energy and Industrial Strategy (BEIS) as part of the Government Chemist Programme 2017–2020, and another (LF) acknowledges support from the Department for the Environment, Food and Rural affairs to provide the freedom to allow work on this book alongside our day jobs. Opinions expressed in the book remain entirely our own. On a personal note, we would like to mention and thank our spouses (Sarah, Tony and Maria) and our families for their support and encouragement; they have graciously tolerated long hours over many months during which we were preoccupied working on the book. Malcolm Burns, Lucy Foster and Michael Walker

Introduction Malcolm Burnsa, Lucy Fosterb and Michael Walkera a

LGC Limited, Queens Road, Teddington, TW11 0LY, UK; bDepartment of the Environment, Food and Rural Affairs, 2 Marsham Street, London, SW1P 4DF, UK

The global food system in the 21st century faces considerable challenges:    “The needs of a growing world population will need to be satisfied as critical resources such as water, energy and land become increasingly scarce. The food system must become sustainable, whilst adapting to climate change and substantially contributing to climate change mitigation. There is also a need to redouble efforts to address hunger, which continues to affect so many.”    Professor Sir John Beddington, former Government Chief Scientist, 2011, Foresight: Future of Food and Farming1    We face unprecedented change in the way we need to grow, produce and manufacture food to ensure we have a sustainable, resilient and secure supply for future generations. Our supply chain must also sustain consumer confidence if we are to grow and maintain a competitive and thriving food industry. Knowing the ingredients of a particular food, how it was produced, where it comes from and that it is correctly labelled, are a key part of assuring the integrity of the food system. These are the central elements of food authenticity. It is also essential that we have test methods to assess the authenticity of foods – the expertise of the contributors to this book has led to the development of many such methods in use today.   Food Chemistry, Function and Analysis No.16 DNA Techniques to Verify Food Authenticity: Applications in Food Fraud Edited by Malcolm Burns, Lucy Foster and Michael Walker © The Royal Society of Chemistry 2020 Published by the Royal Society of Chemistry, www.rsc.org

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Why is DNA of such importance as a marker of food authenticity? Because food authenticity often turns on the question of what a food is – its nature or substance† and how it is described. DNA (Box 1.1) provides an unequivocal answer to these questions. DNA-­based methods to determine the authenticity of food were developed over 20 years ago but it was the horse meat incident in 2013 2 that provided the impetus for a major change in the way in which we conduct DNA analysis. Why is food authenticity important? We have an intimate relationship with food going far beyond its nutritive value into cultural and interpersonal meaning. The authenticity of the food we buy and eat is of visceral significance. Given the time, opportunity and experience we can appraise food visually, and by smell and taste. But we must often take authenticity on trust – when the food's description becomes the key. If that trust is betrayed by misdescription on a personal level we feel cheated. Economically there is loss of ‘value for money’, which many can increasingly ill afford.3,4 Loss of trust at a societal level causes reputational damage to individual companies and difficult trading conditions for whole sectors.5 In some situations individual consumers may come to actual harm, as shown by food allergen fatalities caused by, at times fraudulent, substitution.6 Misleading a purchaser about food authenticity is illegal, potentially harmful, penalises the honest trader, and undermines consumer choice and value for money. In general, when driven by financial gain it is food fraud and when particularly serious, and involves intentional dishonesty detrimentally affecting the safety or authenticity of food, it is food crime7 (Box 1.2). Thus, this book takes the analyst on a ‘food forensics’ journey, setting out how DNA technology, originating in criminology, has paved the way for the successful application of the sophisticated analytical tools we have today to verify food authenticity. It describes the range of tools available, how they have evolved, and how analytical confidence in these methods has been developed. It also describes the practical challenges associated with the application of DNA tools for ‘fit for purpose’ analysis for industry and enforcers alike. Some of these challenges, and the step changes in science development to tackle them, are brought to life through a series of case studies. More broadly, this book provides a European Union (EU) perspective on how authenticity methods and approaches are being harmonised and standardised, drawing heavily on the ‘Genetically Modified Organisms (GMO) story’; and charts EU cooperation on food integrity research over the past 20 years. It also provides an international insight on issues such as food fraud and verifying authenticity of traditional medicines. Finally, looking forward, some of the exciting new developments in DNA analysis and how these are paving the way for future ways to tackle food fraud are explored. †

 he terms ‘nature’, ‘substance’ (and ‘quality’) applied to food have a special significance T in food law, see Chapter 10.

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Box 1.1 DNA (Molecular biologists may skip this…) Nucleic acids were first isolated from cell nuclei over 100 years ago. DNA, deoxyribonucleic acid, is found in virtually every plant, micro-­organism and animal cell, with the exception of mature human red blood cells, which lose their nuclei as they grow. Chromosomes, in the nucleus of each cell and only visible (microscopically) when the cell is dividing, are thread-­like structures of DNA tightly coiled many times around supporting proteins (e.g. histones).8 Genetic information in segments of the chromosomes called ‘genes’ carry instructions from one generation to the next for the growth, development and functioning of the living organism. Genes control cell growth and division and code for the biosynthesis of enzymes and other proteins required for cellular function. The information encoded in DNA is translated into protein via ribonucleic acid (RNA). The now famous double helix structure of DNA was elucidated in 1953 by Francis Crick and James Watson based on previous studies and X-­ray diffraction data from Maurice Wilkins and Rosalind Franklin.9 If the helical structure of DNA is regarded simplistically as a ladder (Figure 1.1) the ‘uprights’ are polymeric chains of phosphoric acid esterified with a pentose sugar, deoxyribose‡, and the ‘rungs’ are heterocyclic amine bases§. Two of the bases are substituted purines: adenine (A) and guanine (G) and two are substituted pyrimidines: cytosine (C) and thymine (T)¶. The bases are bonded to the deoxyribose sugar by an N-­glycosidic link hence they are termed nucleosides. The atomic numbering system in the sugar moiety is 1′ to 5′ (1 prime to 5 prime) thus distinguishing these from the atoms in the heterocyclic amine bases. The 5′-­OH group of the sugar portion of the nucleoside esterified with phosphoric acid forms the nucleoside-­5′-­phosphate or nucleotide. In summary, nucleic acids, such as DNA∥ are polymers of nucleotides and DNA has a direction – one end of the nucleic acid polymer has a free hydroxyl at C3′ (called the 3′ end) and the other end has a phosphate residue at C5′ (the 5′ end) (Figure 1.2).

Figure 1.1 Simplified diagram of DNA double helix. Reproduced from ref. 21 with permission from the Royal Society of Chemistry.

(continued)

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Box 1.1  (continued)

Figure 1.2 Showing a labelled section of the DNA molecule. Reproduced from ref. 22 with permission from the Royal Society of Chemistry.

Figure 1.3 Hydrogen bonding in the DNA double helix. Reproduced from ref. 22 with permission from the Royal Society of Chemistry.

While the nucleotides themselves are covalently bonded, Crick and Watson proposed, and it is now well known, that hydrogen bonding between the heterocyclic amine bases plays a large part in the formation and stability of the double helix structure of DNA. Strong attraction is generated between two (A and T**) or three (C and G††) pairs of atoms (Figure 1.3). Thus two complementary single strands of DNA form the double stranded molecule on which genetic information is inherent. Conventionally, the symbolic shorthand notation reflects this, for example a base pair sequence might be written as:

5′ CCCGAATTCCGC…3′ 3′ GGGCTTAAGGCG…5′

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Box 1.1  (continued) These molecular geometries and hydrogen bonding energy profiles result in the self-­assembly, replication and amplification properties of DNA. Further Reading H. Beyer and W. Walker, Organic Chemistry, A Comprehensive Degree Text and Source Book, ed. and Trans., Douglas Lloyd, Albion Publishing, Chichester, 1997, pp. 849–859. P. Madesis, I. Ganopoulos, I. Sakaridis, A. Argiriou and A. Tsaftaris, Advances of DNA-­based methods for tracing the botanical origin of food products, Food Res. Int., 2014, 60, 163–172. ‡

‘Deoxy’ signifying that one of the sugar hydroxyl groups has been replaced by hydrogen. For example the lone pair of electrons on the –NH of adenine and guanidine feed into the heterocyclic ring to increase electron density and hence basicity on the other nitrogens. ¶ The symbols A, G, C and T may refer to nucleosides, deoxynucleosides, or nucleotides when specifying base-­pairing or nucleotide sequences. ∥ And RNA, where thymine is replaced by the pyrimidine uracil. **=N–H⋯O= and ≡N⋯H – N= [⋯ Indicates hydrogen bonding]. †† =O⋯H–N=, =N–H⋯N≡ and =N–H⋯O=. §

Box 1.2  Food authenticity, adulteration, fraud and crime Concerns that food should be true to its description are not new – food has probably been adulterated since trade commenced.10 ‘Adulteration’ has been the traditional concept to describe food that is unsafe or misdescribed. Scientific appraisal of food began in earnest in the latter half of the 19th century11 when major advances were made in food science, often by researchers charged with upholding newly introduced food law, in the UK the Public Analysts.12–16 False claims about any of the properties of marketed food have been regarded as fraud at least since the 19th century17 and food fraud is now understood to involve any dishonest act or omission relating to the sale or preparation of food intended for personal gain or to cause loss to another party. Economically motivated adulteration is another frequent phrase applied. However, the concept of ‘food crime’ was largely introduced by Professor Chris Elliott during his review18 into the integrity and assurance of food supply networks in the aftermath of the horse meat scandal. Although anything other than authentic food is illegal there is a gradation of seriousness (Figure 1.4) and the National Food Crime Unit (NFCU) of the Food Standards Agency (FSA) and the Scottish Food Crime and Incidents Unit, (SFCIU), define food crime as “… dishonesty in food production or supply, which is either complex or results in serious harm to consumers, businesses or the public interest”.19

Figure 1.4 Gradation of seriousness of food illegality. (continued)

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Box 1.2  (continued) The NFCU further identify20 seven techniques as the main methods through which food crime can be committed:    1. theft – dishonestly appropriating food, drink or feed products in order to profit from their use or sale 2. unlawful processing – slaughtering or preparing meat and related products in unapproved premises or using unauthorised techniques 3. waste diversion – unlawfully diverting food, drink or feed meant for disposal, back into the supply chain 4. adulteration – reducing the quality of food by including a foreign substance, in order to lower costs or fake a higher quality 5. substitution – replacing a food or ingredient with another substance that is similar but inferior 6. misrepresentation – marketing or labelling a product to wrongly portray its quality, safety, origin or freshness 7. document fraud – includes the making, use and possession of false documents with the intent to sell, market or otherwise vouch for a fraudulent or substandard product.

We trust you will enjoy reading the book, perhaps learn from it, and if you spot any (inevitable) errors please let us know. Please also note that any views expressed are those of the editors or chapter authors personally and not those of the Royal Society of Chemistry, Government Chemist, the Department for Environment, Food and Rural Affairs, the Department for Business, Energy and Industrial Strategy or chapter authors' institutions.

References 1. The Future of Food and Farming, Challenges and choices for global sustainability, https://assets.publishing.service.gov.uk/government/ uploads/system/uploads/attachment_data/file/288329/11-­546-­future-­of-­ food-­and-­farming-­report.pdf, Last accessed March 2019. 2. M. J. Walker, M. Burns and D. T. Burns, Horse meat in beef products— species substitution 2013, J. Assoc. Public Anal., 2013, 41, 67–106. 3. M. Boeri, H. Brown and A. Longo, The implications across Europe of the ‘horse meat scandal’ on the monetary value of meat authenticity and food safety in ready to heat lasagne: evidence from six countries, in EAAE 2014 Congress ‘agri-­food and rural innovations for healthier societies, 2014 August, 26–29. 4. V. Bindt, Costs and Benefits of the Food Fraud Vulnerability Assessment in the Dutch Food Supply Chain, 2016, http://edepot.wur.nl/390258, Accessed 31.12.2018. 5. C. Barnard and N. O'Connor, Runners and riders: the horsemeat scandal, EU law and multi-­level enforcement, Camb. Law J., 2017, 76(1), 116–144. 6. M. H. Gowland and M. J. Walker, Food allergy, a summary of eight cases in the UK criminal and civil courts: effective last resort for vulnerable consumers?, J. Sci. Food Agric., 2015, 95(10), 1979–1990.

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7. Food Crime, How we define food crime and how to report it, https:// www.food.gov.uk/safety-­hygiene/food-­crime, Accessed April 2019. 8. U.S. National Library of Medicine, Help Me Understand Genetics, An introduction to fundamental topics related to human genetics, including illustrations and basic explanations of genetics concepts, https://ghr. nlm.nih.gov/primer, accessed 30th December 2018. 9. R. Dahm, Friedrich Miescher and the discovery of DNA, Dev. Biol., 2005, 278(2), 274–288. 10. P. Shears, Food fraud – a current issue but an old problem, Br. Food J., 2010, 112(2), 198–213. 11. H. Deelstra, D. T. Burns and M. J. Walker, The adulteration of food, lessons from the past, with reference to butter, margarine and fraud, Eur. Food Res. Technol., 2014, 239(5), 725–744. 12. D. T. Burns, Alexander Wynter Blyth (1844–1921). A pioneering and innovative Public Analyst, J. Assoc. Public Anal., 2007, 35, 17–29. 13. D. T. Burns, Sir Charles Alexander Cameron (1830–1921) Dublin's Medical Superintendent, Executive Officer of Health, Public Analyst and Inspector of Explosives, J. Assoc. Public Anal., 2009, 37, 14–39. 14. D. T. Burns, Robert Rattray Tatlock (1837–1934) Public Analyst for Glasgow, J. Assoc. Public Anal., 2011, 39, 38–43. 15. P. Clare and M. Clare, The life and times of Alfred Henry Allen Sheffield's first Public Analyst, J. Assoc. Public Anal., 2012, 40, 39–59. 16. D. T. Burns and M. J. Walker, Edmund Albert Letts (1852–1918)–A Pioneer Environmental Analytical Chemist and his Association with Official Analytical Posts in Ulster, J. Assoc. Public Anal., 2015, 43, 13–26. 17. See for example Anon, Offences against the Sale of Food and Drugs Act, Analyst, 1876, 1, 174. 18. Elliott Review into the Integrity and Assurance of Food Supply Networks – Final Report, A National Food Crime Prevention Framework July 2014, https://www.gov.uk/government/publications/elliott-­review-­into-­the-­ integrity-­and-­assurance-­of-­food-­supply-­networks-­final-­report, accessed 31.12.2018. 19. Food Standards Agency, Food Standards Scotland, 2016, Food Crime Annual Strategic Assessment, 2016 Baseline, https://www.food.gov.uk/ sites/default/files/media/document/fsa-­food-­crime-­assessment-­2016. pdf, accessed 31.12.2018. 20. Food Standards Agency, National Food crime Unit, 2018, https://www. food.gov.uk/safety-­hygiene/food-­crime, accessed 31.12.2018. 21. A. P. Damant for the Analytical Methods Committee, AMC Technical Briefs, 2009, AMCTB 35, https://www.rsc.org/images/dna-­technical-­ brief-­35_tcm18-­214870.pdf, accessed April 2019. 22. R. J. Simmonds, Chemistry of Biomolecules, Royal Society of Chemistry, Cambridge, 1992.

Contents Chapter 1 The Role of DNA Analysis in the Determination of Food Authenticity  S. B. Primrose

1.1 A Brief History of Food Adulteration  1.2 Food Fraud in the 21st Century  1.3 Challenges in Detecting Food Misdescription   and Fraud  1.4 The Role of DNA in Food Authenticity Determination  1.5 Application of DNA-­based Analytical Methods to   Different Commodities and Food Authenticity   Problems  1.6 Pushing the Boundaries: Specialist Techniques for   Breed or Variety Identification and Determination   of Geographical Origin  1.7 Fitness for Purpose  1.8 Final Comments  References  Chapter 2 Forensic DNA – Criminal and Paternity Methods and Applications – How Can This Help in Verifying Food Authenticity?  Victoria Moore



2.1 Some History  2.2 Applying the Forensic Code of Conduct  2.3 Key Issues – Complexities Within Forensic   Regulatory Bodies and Associations 

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2.4 Key Issues – Databases and Communication  2.5 Document Recording 2.5.1 Receipt of Materials  2.5.2 Examination, Standard Operating   Procedures and Quality Control  2.6 Conclusion  References 

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Chapter 3  DNA Extraction from Food Matrices  Timothy Wilkes

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3.1 Introduction  3.2 Factors Influencing the Choice of Extraction   Methodology 3.2.1 Sample Source and Processing  3.2.2 Sample Collection and Storage  3.2.3 Homogenisation  3.2.4 Presence of Contaminants  3.3 General DNA Extraction Methods  3.3.1 Phenol–Chloroform Extractions  3.3.2 Detergent and Protease-­based Extractions  3.3.3 Solid-­phase Extraction Methods  3.3.4 Concentrating DNA Post-­extraction  3.4 DNA Extraction Methods Frequently Employed   with Food and Feed Samples  3.5 Determining DNA Quantity and Purity  3.5.1 Quantification of DNA  3.5.2 Evaluation of DNA Purity  3.5.3 RNA Contamination  3.5.4 Non-­nucleic-­acid Contamination  3.5.5 Evaluation of a DNA Sample's Integrity  3.5.6 Determining the Presence of Inhibitory   Compounds  3.6 Concluding Remarks  Acknowledgements  References 

30 30 30 30 31 33 34 34 35 36 37 39 39 42 44 44 44 45 46 47 47

Chapter 4  “DNA Techniques” Case Study: Isothermal Approaches  G. Nixon

50



50 51 51 51

4.1 Introduction  4.2 Main Isothermal NAA Technologies  4.2.1 Cross Priming Amplification (CPA)  4.2.2 Helicase-­dependent Amplification (HDA)  4.2.3 Loop-­mediated Isothermal Amplification   (LAMP) 

52

Contents



xxi

4.2.4 Recombinase Polymerase Amplification   (RPA)  4.3 PCR vs. Isothermal Technologies  4.4 Food Applications  4.4.1 Meat Speciation  4.4.2 GMOs  4.4.3 Allergens  4.5 Future View  4.6 Conclusion  Acknowledgements  References  Chapter 5 Digital Polymerase Chain Reaction (dPCR) – General Aspects and Applications  S. Pecoraro



5.1 Introduction  5.2 General Properties of dPCR  5.3 dPCR Platforms  5.4 Assumptions for Absolute DNA Quantification   with dPCR  5.5 Applications of dPCR in Food Analysis  5.6 Future Aspects  References  Chapter 6 UK Food Authenticity Programme – The Analytical Tool Box  L. H. Foster and S. B. Primrose



6.1 Introduction: Drivers and Rationale for the   Programme  6.2 Policy Context: Industry, Enforcement and   Consumer Trust  6.3 Technical Challenges Affecting Policy   Development and Food Law Enforcement  6.4 The Analytical Toolbox  6.4.1 Chemical Methods  6.4.2 Physical Methods  6.4.3 Proteomic-­based Methods  6.4.4 DNA-­based Methods  6.5 Upskilling: Knowledge Transfer of Government-­  funded DNA Techniques to Support Food Law Enforcement  6.6 Forward Look and Future Challenges  6.6.1 Collaboration – a Global Response to   Food Fraud 

54 55 55 55 57 57 57 58 58 59 63 63 64 65 66 67 67 68 70

70 71 74 74 75 76 76 77 79 79 80

Contents

xxii





6.6.2 Future Tools to Tackle Food Fraud  6.6.3 Food Labelling and Informed Choice  6.7 Conclusion  Acknowledgement  References 

Chapter 7  Fitness for Purpose of DNA-­based Analytical Methods  S. B. Primrose and L. H. Foster







7.1 Introduction  7.2 Fitness for Purpose  7.2.1 What is ‘Fitness for Purpose?’  7.3 Challenges for Fitness for Purpose for   Authenticity  7.4 Sampling and Extraction  7.5 Qualitative Versus Quantitative Analysis  7.6 Reference Materials  7.7 Validation  7.8 Measurement Uncertainty  7.9 How is Fitness for Purpose Achieved?  7.10 Full Validation  7.11 Conclusions  References 

Chapter 8 GMO Detection and Identification Using Next-­generation Sequencing  Marie-­Alice Fraiture, Nina Papazova, Kevin Vanneste, Sigrid C. J. de Keersmaecker and Nancy H. Roosens

8.1 The Current Landscape of GMO Detection  8.2 Applying NGS to GMO Detection: Current   Approaches  8.3 Challenges for the Detection and Characterization   of GMOs Using NGS Related to the Host  8.4 Conclusion and Perspectives  References 

Chapter 9  A Perspective on Quantitative DNA Approaches  Malcolm Burns

9.1 Introduction: The Requirement for Accurate   Quantitation of Food Samples  9.2 Recommendations for Methods for Accurate   Quantitation of Food Samples  9.3 Approaches for Quantitative Estimation of   Food Samples Using DNA 

80 81 81 82 82 86 86 87 87 88 88 89 91 91 91 92 94 94 94 96

96 97 102 103 103 107

107 109 111

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xxiii

9.3.1 Choice of Target for Quantitative Estimation  9.3.2 Choice of Method for Quantitative   Estimation  9.3.3 Expression Units and Measurement Scales  9.4 Ensuring Fitness for Purpose for Accurate   Quantitation of Food Samples  9.5 Summary and Future Perspectives on   Quantitative DNA Approaches  Acknowledgements  References 

111 111 113 115 116 117 117

Chapter 10  DNA in Food and Feed Law  Nigel Payne

120



120 121 121 124 124 125 125 125 126 127 128 129 130 130 130 131



10.1 Introduction  10.2 Food  10.2.1 Framework Law  10.2.2 Food – Subordinate Law  10.2.3 Protected Designations  10.3 Other Authenticity Issues  10.3.1 Honey  10.3.2 Herbs and Spices  10.4 Allergens in Food  10.5 Microbiological Safety of Food  10.6 Genetically Modified Organisms – GMOs  10.6.1 GM Rice and Rice Products  10.7 Organic Food and Feed  10.8 Protection of Vegan, Halal and Kosher Food  10.9 Investigation of Food “Complaints”  10.10 Animal Feeding Stuffs  10.11 Legal Provisions for Official Controls of   Food and Feed  10.12 Taking of Samples  10.13 Interpreting Results in a Legal Context  10.14 EU Exit  References 

131 132 133 134 134

Case Studies 

139

Chapter 11  Harmonising DNA Methods – The GMO Story  Hendrik Emons

141



141 142 143 144 145 145

11.1 Introduction  11.2 The Harmonisation Challenge  11.3 The Network Approach  11.4 Achievements and Current Status  Acknowledgements  References 

Contents

xxiv

Chapter 12  Metrology of DNA Approaches  Mojca Milavec, David Dobnik, Alexandra Bogožalec Košir and Jana Žel

147



147 148



12.1 Introduction  12.2 Key Comparison Studies  12.3 Partition Volume Variability – a Critical Factor Influencing the Accuracy of Absolute   Quantification by Digital PCR  12.4 Conclusions  References 

Chapter 13 The Almond and Mahaleb Allergen Story – PCR Resolution of Live Incident Investigations  Michael Walker and Malcolm Burns

13.1 Introduction  13.2 Almond and the Prunus Family  13.3 Sample Authentication  13.3.1 Real Time PCR Assay Development  13.4 Results  13.4.1 Referred Cumin Sample  13.4.2 Referred Paprika Sample  13.5 Conclusions  Acknowledgements  References 

Chapter 14 Food Fraud Prevention – Selecting the Right Test, Method, and Sampling Plan  John Spink

14.1 Introduction  14.2 Food Fraud Overview  14.3 Criminology and Food Fraud Prevention  14.4 Case Study: Horsemeat Incident  14.5 Conclusion  References 

150 151 152 154 154 155 155 156 157 157 157 158 159 159 162 162 163 163 164 166 166

Chapter 15  Meat Speciation  Maria Karczmarczyk

169



169 170 170 171 171 171

15.1 Comparison Between PCR and ELISA  15.2 Qualitative PCR Testing  15.3 Quantitative PCR Testing  15.3.1 Low DNA Content Samples – Dairy, Gelatine  15.3.2 Effects of Food Processing  15.3.3 Interference – Inhibition 

Contents



xxv

15.3.4 Limit of Detection (LOD)  15.4 Proprietary Methods – Considerations and   Validation Approaches  15.5 Reporting  15.6 New Developments  References 

Chapter 16 The Horse Meat Scandal – The European Analytical Response  Marien Aline, Fumière Olivier, Debode Frédéric, Hulin Julie and Berben Gilbert

16.1 Introduction  16.2 Material and Methods  16.2.1 Real-­time PCR Analysis  16.2.2 Specificity of the Horse PCR Method  16.2.3 Sensitivity of the Horse PCR Method  16.2.4 Robustness of the Horse PCR Method  16.2.5 Development of a Semi-­quantitative   Method for the Detection of Horse Meat  16.3 Results  16.3.1 Evaluation of the Performance of the   Horse PCR Method  16.3.2 Development of a Semi-­quantitative   Method for the Detection of Horse Meat  Acknowledgements  References 

172 173 174 174 175 177

177 178 178 179 180 180 181 181 181 182 185 185

Chapter 17 Horse Meat: Technical Appeals and Court Action  Michael Walker

189



189 190 190 191 192 192 194 194 194



17.1 Introduction  17.2 Technical Appeals  17.2.1 Technical Approach  17.2.2 DNA  17.2.3 ELISA  17.2.4 Interpretation  17.3 Prosecutions  17.3.1 Bulgarian Sausage  17.3.2 Abattoir with no Horse Traceability  17.3.3 Frozen Blocks of Meat and ‘Bargain’   Beef Burgers with Horse  17.3.4 Heart in Burgers  17.3.5 The Netherlands  17.3.6 France  17.4 Conclusions  References 

195 196 196 196 197 197

Contents

xxvi

Chapter 18  Durum Wheat and Pasta Authenticity  Gordon Wiseman

200



200 201 201



18.1 Introduction and Regulations  18.2 Case Study  18.2.1 Is T. aestivum Present in the Pasta?  18.2.2 qPCR Confirmation and Quantification   of the T. aestivum  18.2.3 Analytical Uncertainty  18.3 Conclusions  Acknowledgement  References 

202 204 205 205 206

Chapter 19  The Authenticity of Basmati Rice – A Case Study  Mark Woolfe and Katherine Steele

207



207



19.1 Introduction  19.2 Methodology Development to Authenticate   Basmati Rice  19.2.1 Basmati Rice Variety Authentication and Quantification  19.3 Food Standards Agency's Basmati Rice   Survey  19.3.1 Basmati Rice Variety Identification  19.3.2 Quantitative Measurement of   Non-­basmati Varieties  19.3.3 Follow-­up Action of the Survey  19.4 Further Development of the Methodology  19.4.1 Basmati Variety Identification  19.4.2 Quantitative Determination of   Non-­basmati Varieties in Basmati Rice  19.4.3 Lab-­on-­a-­chip Approach to Basmati Rice Authenticity  19.5 Postscript to Basmati Rice Methodology  Acknowledgements  References 

Chapter 20 Horse Meat: The International Collaborative Trial of the Real-­time PCR Method for the Quantitation of Horse DNA  Malcolm Burns and Lucy Foster

20.1 Food Fraud  20.2 The 2013 Horse Meat Issue  20.3 Challenges in Meat Quantitation 

208 208 209 209 209 211 211 211 213 214 216 216 217

219 219 220 220

Contents



xxvii

20.4 Development of a Real-­time PCR Method for the Quantitation of Horse DNA  20.5 International Collaborative Trial of the Real-­time   PCR Method for the Quantitation of Horse DNA  20.6 Summary  Acknowledgements  References 

Chapter 21 Standardization of DNA-­based Methods for Food Authenticity Testing  L. Grohmann and C. Seiler

21.1 Introduction  21.2 CEN and ISO – Activities and Projects  21.2.1 CEN WS/86 – Authenticity in the Feed and   Food Chain – General Principles and Basic Requirements  21.2.2 CEN Food Authenticity Coordination Group   (FACG) and a New Technical Committee   on Food Authenticity  21.2.3 CEN/TC 275/WG 11 ‘Genetically Modified Organisms and Species Analysis’  21.2.4 ISO/TC 34/SC 16 ‘Horizontal Biomarker   Analysis’  21.3 Discussion and Outlook  21.4 Conclusion  Acknowledgement  References 

Chapter 22 Authentication of Chinese Traditional Medicine by DNA Analysis  Foo Wing Lee, Olive Tin Wai Li and Winnie Wing Yan Chum

22.1 Introduction  22.2 Reliability of DNA Methods for Species   Identification  22.3 Selection of DNA Markers  22.4 Bioinformatics Analysis  22.5 Demo Analysis  22.6 Example Cases  22.6.1 Authentication of Cordyceps  22.6.2 Authentication of Oviductus Ranae  22.7 Conclusion  Acknowledgements  References 

221 222 224 224 225 227 227 228 228 229 229 232 233 233 234 234 235 235 236 237 238 243 245 247 250 251 251 251

Contents

xxviii

Chapter 23  DNA Point of Use Applications  Michael Walker, Malcolm Burns and Lucy Foster

255



255 256 256 257 257 257 258 258 259 261 261 261

23.1 Introduction  23.2 PoU in Criminal DNA Profiling  23.2.1 ParaDNA™  23.2.2 RapidHIT by IntegenX  23.3 PoU in Point of Care Diagnostics  23.3.1 Genie II/III™ by OptiGene  23.3.2 QuantuMDx Q-­POC™  23.3.3 Biocartis Idylla™  23.4 Discussion  23.5 Conclusions  Acknowledgements  References 

Chapter 24  Commercial DNA Testing  Barbara Hirst, Lourdes Fernandez-­Calvino and Thomas Weiss

264



264 265 266 267 268 268



24.1 Introduction  24.2 Expectations from Commercial Clients  24.3 Are Expectations Always Deliverable?  24.4 DNA Approaches  24.5 Overview of Commercial DNA Tests  24.5.1 DNA Detection Methods  24.6 Comparison of Commercial DNA Tests and   In-­house Methods  24.7 Challenges in the Development of In-­house   DNA Test Methods  24.8 Reporting PCR Results  References 

Chapter 25 EU Food Integrity and Joining up the Landscape (EU Perspective)  Elena Maestri and Nelson Marmiroli

25.1 The Turning Point in EU Food Studies  25.2 Research on Food in the EU  25.2.1 The Beginning  25.2.2 Research at the Turn of   the Century  25.2.3 The Advent of Food Integrity  25.2.4 Research in FP7  25.2.5 Horizon 2020  Acknowledgements  References 

276 277 278 280 283 283 284 285 289 292 293 294 294 294

Contents

xxix

Chapter 26  The Food Authenticity Network  Selvarani Elahi, Stephen Ellison and Mark Woolfe

296



296



26.1 Background  26.2 Creation of the Food Authenticity   Network  26.2.1 Network Structure  26.3 Centres of Expertise (CoE) for Food   Authenticity Testing  26.4 Authenticity Research and Methods  26.5 Training  26.6 Discussions and Forums  26.7 Policy and Law  26.8 Quality  26.9 Relevant Organisations  26.10 Latest News and Events  26.11 Food Fraud Mitigation  26.12 Social Media Platforms  26.12.1 Twitter  26.12.2 LinkedIn  26.13 Growth  26.14 Impact  26.14.1 Membership  26.14.2 Member Surveys  26.14.3 Analytics Data  26.15 International Recognition  26.16 Global Network  26.17 Conclusions  References 

297 298 299 300 300 300 301 301 301 301 302 302 302 303 303 303 303 305 307 307 309 309 310

Chapter 27  A Vision for the Future  L. H. Foster, M. Burns and M. Walker

311



311 312



27.1 Introduction  27.2 Changing World  27.3 Food Fraud Prevention – Technological   Opportunities  27.4 Analytical Developments and Future Needs  27.5 Labelling and the Consumer  27.6 Conclusions  References 

Subject Index 

312 313 315 315 316 318

Chapter 1

The Role of DNA Analysis in the Determination of Food Authenticity S. B. Primrose* 21 Amersham Road, High Wycombe, Buckinghamshire HP13 6QS,   United Kingdom *E-­mail: [email protected]

1.1  A Brief History of Food Adulteration Friedrich Accum was a German chemist who came to London in 1793 and established himself as an analyst. His interests included keeping processed food free from dangerous additives. In 1820 he published a best-­selling book entitled ‘A Treatise on Adulterations of Food and Culinary Poisons’.1 In this book he describes practices such as colouring red cheese and confectionary with red lead and mercuric sulphide, using strychnine instead of hops in the production of beer, whitening bread flour with alum and chalk and extending bread loaves with plaster of Paris and sawdust. Other practices included boiling spent tea leaves with Prussian Blue (ferric ferrocyanide) and sheep dung, which were dried and re-­sold. Arthur Hill Hassall (a London physician) began a major investigation into food adulteration in the 1850s. One of his early successes used microscopy to demonstrate the adulteration of coffee with chicory. Hassall's work, together with Henry Letheby, the Medical Officer of Health for London, led to the   Food Chemistry, Function and Analysis No.16 DNA Techniques to Verify Food Authenticity: Applications in Food Fraud Edited by Malcolm Burns, Lucy Foster and Michael Walker © The Royal Society of Chemistry 2020 Published by the Royal Society of Chemistry, www.rsc.org

1

2

Chapter 1

introduction of the Adulteration of Food and Drugs Act of 1860. This Act was revised in 1872 and again in 1875. The Sale of Food and Drugs Act 1875 is widely regarded as a turning point in the regulation of food, introducing key concepts such as that food must be of the ‘nature’ or ‘substance’ or ‘quality’ demanded by the purchaser. It included, as a duty of local government, the appointment of a certain type of scientist, the Public Analyst – a key Hassall recommendation – to provide the underpinning analytical data and its interpretation for the enforcement of the provisions of the Act. The Society of Public Analysts was formed (now known as the Association of Public Analysts). Today, one of the key tasks of public analysts in the UK and Ireland is to ensure the safety and correct description of food by testing for compliance with legislation. As described in Chapter 10, legislation that relates to the authenticity of food remains in place today. In the UK, the Food Safety Act 1990 prohibits ‘falsely or misleadingly describing or presenting food’. Regulation (EU) No.1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers, (implemented in the UK as the Food Information Regulations 2014, No.1855) as amended and with devolved equivalents specifies what information must be given with marketed food. For pre-­packed food the required information includes a list of the individual components, the amounts present (quantitative declaration of ingredients or QUID) and, where appropriate, the country of origin of key ingredients. Inherent within this regulation is that, with very limited exceptions, pre-­packed food should not include any undeclared ingredients. Despite this legislation, food adulteration and other forms of food fraud continues to exist. INTERPOL and Europol have undertaken joint operations targeting counterfeit and sub-­standard foodstuff and beverages. For example, operation OPSON V in 2015, involving 57 countries, where the operation seized over 10 000 tonnes of food and one million litres of drink which was either counterfeit or sub-­standard.

1.2  Food Fraud in the 21st Century Consumers expect that the food they buy is labelled correctly but, as the example above shows, food fraud is prevalent despite the existence of relevant legislation. Food fraud can broadly take three forms: adulteration, substitution and mis-­description. Adulteration includes the addition of undeclared ingredients. Examples include adding horsemeat or offal to beef in processed foods, adding illegal colourants to improve the appearance of food and adding water to frozen food to increase its weight. Other examples of food adulteration are given in Box 1.1. Substitution involves replacement of one ingredient by a similar or cheaper one. Examples include using whiting or pollack in place of cod, bonito in place of tuna or sea trout to replace salmon. A survey by Oceana found this

The Role of DNA Analysis in the Determination of Food Authenticity

3

Box 1.1  Some examples of food adulteration. ●●Mixing long-­grain rice with Basmati rice ●●Mixing cow's milk with buffalo milk before producing buffalo mozzarella

cheese

●●Adding common wheat to durum wheat pasta labelled as 100% durum wheat ●●Extracting soluble coffee from beans mixed with skins and husks ●●Adding cheaper vegetable oils to named higher-­value vegetable oils ●●Adding glycerol to wine to extend body ●●Adding mandarin or tangerine juice to orange juice to improve colour ●●Painting green olives with copper sulphate to improve colour

Table 1.1  Examples  of misdescription of food. Type of Misdescription

Examples

Non-­declaration or   false declaration of   processes

●● Labelling previously frozen meat or poultry as fresh ●● Failure to declare that food has been irradiated ●● Failure to declare that juice has been made from

concentrate

●● Including animal-­derived ingredients in vegetarian

Over-­declaration   of a quantitative   ingredient False claims regarding geographical origin   or method of production

meals

●● Including hydrolysed protein as part of the meat

content

Labelling South American beef as British beef Declaring farmed fish as ‘wild’ Labelling conventionally produced food as organic Claiming that extra virgin olive oil is from a particular geographical region ●● Using bovine or porcine gelatine hydrolysates and claiming these are of chicken or fish origin ●● ●● ●● ●●

type of fraud to be widespread in restaurants in the USA and Europe.2 Similar studies aimed specifically at sushi restaurants found substitution varying from 10% in the UK to 47% in the USA.3,4 Misdescription of food takes several forms and usually relates to the method of production, the geographical origin of the food or the amounts of key ingredients used. Some representative examples are given in Table 1.1. In the USA, food fraud is treated primarily as a food safety issue. In contrast, in Europe food fraud is viewed as a breach of food labelling law with food safety being covered by separate legislation. Nevertheless, there is a clear link between food fraud and food safety. Examples include the US Oceana surveys,2 where most of the fish labelled as tuna was in fact escolar, which can cause digestive problems. Another example of a safety issue related to adulteration is the use of known carcinogens (illegal dyes), such as Sudan 1, to improve food colouration and increase the price of spices such as chilli powder.

4

Chapter 1

There are a number of drivers of food fraud. Two key ones are price pressures on food suppliers and criminal activity. Low profit margins for food manufacturers create cost pressures if raw materials or utilities increase in price. This can potentially create opportunities for food fraud by reducing costs via adulteration or substitution of key ingredients. Criminal involvement usually is associated with high value or premium products where huge profits can be made by passing off a cheaply produced or inferior quality product as a superior version. Such criminal activity operates at all levels of food production; and is attractive due to its perceived low penalties, for example, compared with drugs trafficking.† Further discussion on food fraud is provided in Chapter 14.

1.3  C  hallenges in Detecting Food Misdescription and Fraud There are five main challenges in developing methods to determine the authenticity of food. Each generates a requirement that the methods are fit for purpose. The first challenge is the choice of analytical method. The range of misdescription and fraud described in the previous section means that no single methodology will suffice. As discussed elsewhere in this book, it may be necessary to use multiple methods. The second challenge is finding a marker or markers that characterises the food, one of its ingredients, the adulterants in question, or its processing, production or geographical origin. The marker has to be specific, its natural variation must be limited and well-­characterised and be measured accurately. The third challenge relates to the variety of matrices that exist in foods. No two processed foods contain exactly the same ingredients. A method developed for one food may not be suitable for use with another. If processed foods are cooked, any markers of interest may be degraded or destroyed completely. The fourth challenge is that most investigations of food adulteration are linked to a legal requirement, standard or guidance. As such, the interpretation of the results must be made in the light of analytical uncertainty, natural variation and any tolerance permitted by the requirements. That is, the conclusion reached must be beyond reasonable doubt. The final challenge relates to the requirement for authentic samples and/ or certified reference materials for the development and evaluation of the method. The difficulties in obtaining authentic samples cannot be over-­ emphasised. Often, once sourced, there is no central repository for their maintenance and supply to analysts. Further discussion on drivers and challenges for food fraud adulteration and its determination is provided in Chapter 6. †

https://www.foodmanufacture.co.uk/Article/2011/11/23/Criminals-­drop-­drugs-­for-­food-­fraud.

The Role of DNA Analysis in the Determination of Food Authenticity

5

1.4  T  he Role of DNA in Food Authenticity Determination All cellular organisms contain DNA. The differences between organisms i.e. anatomical, physiological etc. ultimately resides in their DNA sequence. The more evolutionarily distant two species are, the greater will be the differences in their DNA sequences. However, even when two individuals are closely related, e.g. family members, there are sufficient differences at the DNA sequence level for each individual to be recognised. This is the basis  of DNA profiling used in police and paternity investigations and which came to prominence in the 1980s. In the early 1990s the UK Ministry of Agriculture, Fisheries and Food (MAFF) began funding research to develop analytical methods to detect food fraud, which included the application of forensic DNA profiling to determine the authenticity of food. DNA analysis is particularly suited to qualitative analysis to answer yes/no questions e.g. has there been substitution of one species with another, which formed the focus of much of the early DNA-­ based methods work. As explored in Chapters 6 and 7, public analysts were not overly familiar with the diversity of emerging new DNA techniques for food testing. This issue was resolved when the Agilent lab-­on-­a-­chip analyser became available resulting in the conversion of these DNA techniques to this easier to use format (Chapter 6). More recently, qualitative analysis has been facilitated by the development of DNA barcoding (www.barcodeoflife.org). Barcoding provided a way of distinguishing and identifying species with a short, standardized gene sequence.5 A 648 bp region of the mitochondrial cytochrome-­C oxidase-­1 (CO1) gene was proposed. This proposal has been adopted internationally for the identification of many animal species. However, in plants there is insufficient variation in this gene to be useful. For plant species identification, two gene regions in chloroplasts, matK and rbcL, are often used, although there is still no agreement on a universal barcode to use for plants. Police and paternity forensic work and food authenticity determination differ because for the latter there is often a requirement for quantitative analysis to authenticate foods. This requirement stems from the need to distinguish between adventitious contamination and deliberate fraud. Historically it was accepted that, as a rule of thumb, undeclared species at less than the 5% (w/w) level was adventitious contamination. Following the 2013 horsemeat incident,6 the UK Food Standards Agency set an upper limit of 1% (w/w) for undeclared ingredients with a requirement for explanation if an undeclared ingredient was found to be present at greater than 0.1% (w/w). However, there are exceptions. For example, for EU approved Genetically Modified Organisms (GMOs), food and feed products that contain 20 pg µL−1), inclusion of molecular biology grade glycogen to a final concentration of 0.05–1 µg µL−1 can help improve the level of recovery obtained. Following isolation, the purified DNA should be re-­suspended in a marginally alkaline storage buffer, typically TE buffer, or ultra-­pure water.

3.3.4.2 Concentrating Using a Microfiltration Device A number of low-­bind microfiltration devices are currently available (e.g. the Microcon® 100 centrifugal filter unit). These devices incorporate filtration membranes which allow for the free movement of solvent but restrict the migration of solute molecules. They can provide high levels of recovery (>95%), are ideal for use with dilute DNA solutions (ng mL−1 to µg mL−1) and generate highly reproducible results. Normally, the solution to be concentrated is applied to the filter, which is then subject to centrifugation at a defined g-­force and fixed duration. Excess solvent migrates through the filter while the solute is retained. The filter is then inverted and the centrifugation repeated in order to recover the concentrated solute.

3.4  D  NA Extraction Methods Frequently Employed with Food and Feed Samples In addition to the general methods described above, there are a variety of in-­house methods and commercial kits available which can be used for the extraction of DNA from food and feed samples, which are either universal or matrix-­specific. Examples of methods recommended for use by the European Union Reference Laboratory for GM in Food and Feed (EU-­RL GMFF) are shown in Table 3.1, and example methods that have previously been  used in Department for Environment, Food and Rural Affairs (Defra) funded  food and feed projects are summarised in Table 3.2. Methods based on the  use of CTAB have been extensively used for many of the listed projects. However, only one of the examples listed, the Wizard® magnetic DNA purification system (Promega), has been associated with a binding standard operating

GM Food and Feed (EU-­RL GMFF).a

Source

Method

Reference

Date

Manufacturer

Animal Feed

EURL AP Standard Operating Procedure DNA extraction using the “Wizard® magnetic   DNA purification system for food” kit DNA extraction method for cotton seeds   and grains DNA extraction method from cotton seeds

Binding SOP

29/03/2013

CRLVL24/04XP

20/09/2011

CRLVL13/04XP

14/03/2007

CRLVL02/04XP

28/02/2005

Maize

“CTAB/Wizard” method for DNA extraction from ground maize grain/seed DNA extraction method for maize seeds and grain

CRLVL16/05XP

13/10/2008

Oilseed Rape

Oilseed rape DNA extraction method from seeds

CRLVL06/04XP

11/01/2007

Oilseed Rape

Oilseed rape DNA extraction from seeds

CRLVL26/04XP

07/02/2007

Potato

“CTAB/Microspin” method for DNA extraction   from freeze dried potato tubers “Dellaporta et al.-­derived” method for DNA   extraction from ground rice grains DNA extraction method for ground Soybean

CRLVL14/04XP

07/09/2006

Wizard® magnetic DNA purification system (Promega) CTAB precipitate (in-­house) CTAB/Genomic   20/G Tip (Qiagen) CTAB/Wizard® clean up column (Promega) CTAB precipitate (in-­house) SDS Dellaporta et al.4 (in-­house) CTAB precipitate (in-­house) CTAB/Microspin

CRLVL05/04XP

09/06/2006

CRLVL0109/XP

20/09/2011

“CTAB/Genomic-­tip" method for DNA extraction   from ground sugar beet seeds

CRLVL28/04XP

31/01/2006

Cotton Cotton Maize

Rice Soybean Sugar Beet

38

Table 3.1  Example  nucleic acid extraction methods recommended for use with food by the European Union Reference Laboratory for

SDS Dellaporta et al.4 (in-­house) CTAB precipitate (in-­house) CTAB/Genomic   20/G Tip (Qiagen)

a

Example DNA extraction methods shown in the table can be found at the EU-­RL GMFF website at http://gmo-­crl.jrc.ec.europa.eu/StatusOfDossiers.aspx.

Chapter 3

DNA Extraction from Food Matrices

39

procedure (SOP) (EURL AP Standard Operating Procedure). In addition to being used for the isolation of DNA, the method can also be used for the removal of polymerase chain reaction (PCR) primers, oligonucleotide adaptors and unincorporated label, as well as for the purposes of desalting and buffer exchange.

3.5  Determining DNA Quantity and Purity Post isolation, measurement of DNA concentration and purity is important for many applications in molecular biology. The following section provides information on the theory and practice behind measuring DNA purity and concentration. DNA concentration can be determined with the use of three different methods: (1) optical density (absorbance), (2) agarose gel electrophoresis and (3) fluorescent DNA-­intercalating dyes (Figure 3.5). Two frequently employed methods for determining DNA concentration and purity are the measurement of absorbance for ultraviolet light (UV), which can be determined with use of a UV spectrophotometer and electrophoretic agarose gel analysis. However, more recently, fluorescent DNA-­binding dye methods have become increasingly used to provide accurate quantitative measurements of double stranded DNA concentration. In practice, absorbance values obtained for readings taken from 230 nm to 320 nm are used to provide an indication as to the level of purity of a DNA solution and used to make a judgement call as to the suitability of a sample for use in any downstream molecular biology applications.

3.5.1  Quantification of DNA The value for absorbance at 260 nm (A260) can be used to calculate the concentration of nucleic acids in solution (Figure 3.6). At a concentration of 1 µg mL−1 and for an optical path length of 1 cm, double stranded DNA (dsDNA) has an A260 of 50. However, the measured absorbance value often deviates from this theoretical value due to the presence of DNA secondary structure and a feature known as hypochromicity. The approach is based on the Lambert–Beer law21 which relates the absorption of light to the properties of the material through which the light is travelling. The law states that there is a logarithmic dependence between the transmission of light through a substance and the product of the absorption coefficient of the substance and the distance the light travels through the material (i.e. the path length). However, this relationship breaks down at high concentrations of solute, especially where the solute is highly scattering. For reliable spectrophotometric DNA quantification, it is recommended that a series of dilutions be prepared in order to check the concentration and accuracy of the measurements. As inaccuracies can occur

Table 3.2  Example  nucleic acid extraction methods recommended in DEFRA/FSA project reports for use with food matrices.a Defra ref

Product or kit

Manufacturer

2010

Fish

Q01069

2010 2009 2009

Fish Fruit Juice Meat and Fish

Q01099 Q01114 Q01033

2009 2008

Fruit Juice Cooked Meat

Q01114 Defra/FSA-­Q01084

2008

Hazelnut oil in Olive oil

Defra/FSA-­Q01095

2008

Fruit Juice

Q01111

2008 2007 2007 2003

Meat Bushmeat Pasta Fish

Q01084 Q01109 Q01106 Q01051

2003 2002

Meat Fish

Q01049 Q01006

CTAB–Proteinase K–Wizard® Resin Tepnel Biokits CTAB–Proteinase K–Wizard® Resin Nucleon™ Phytopure™ Midas Genomic–Forensic DNA Kit Genome Star Kit Wizard® Clean Up DNeasy® Tissue Kit GenElute™ Nucleon™ Phytopure™ CTAB–Proteinase K–Wizard® SDS–Proteinase K–Wizard® Chelex® CTAB–Proteinase K–GenElute™ Nucleoplex™ DNA extraction kit Promega Wizard® purification resin Promega Wizard® magnetic purification CTAB–Proteinase K–Wizard® Resin DNeasy® Plant kit Maxwell™16 Wizard® magnetic beads Tepnel magnetic bead extraction DNeasy® kit Wizard® clean up CTAB–Proteinase K Nucleon™ Soft Tissue kit Wizard® Resin + Guanidinium HCl Phenol–Chloroform Wizard® resin with SDS and Guanidinium Cl

Promega Tepnel Promega GE Healthcare Biogene Thermo Promega Qiagen Sigma GE Healthcare Promega Promega Bio-­Rad Sigma Tepnel Promega Promega Promega Qiagen Qiagen Promega Tepnel Qiagen Promega Promega GE Healthcare Promega Sigma/Fisher Promega

a

Example DNA extraction methods shown in the table can be found at the Food Authenticity website http://www.foodauthenticity.uk/research.

Chapter 3

Material

40

Date

DNA Extraction from Food Matrices

41

Figure 3.5  A  flow diagram illustrating the process of determining DNA quantity and purity.

Figure 3.6  An  example of absorption spectra obtained for samples of pure DNA.

Chapter 3

42

where the DNA is not homogeneously suspended, care should be taken to ensure that thorough mixing of the DNA is performed. To improve the accuracy of DNA concentration determination, an allowance for the presence of particulate impurities in the solution needs to be made. This can be achieved by implementing a turbidity adjustment to the A260 measurement which can be derived from the absorbance measurement at 320 nm (A320). The adjustment can be performed using the following formula. Concentration (µg mL−1) = (A260 reading − A320 reading) × dilution factor × 50 µg mL−1 DNA yield can therefore be determined by multiplying the DNA concentration by the purified sample volume using the following formula.

DNA Yield (µg) = DNA Concentration × Total Sample Volume (mL)

3.5.2  Evaluation of DNA Purity Whilst the total mass of DNA that can be isolated from a sample is important, the purity and integrity of that DNA are the fundamental determining factors that determine its suitability for use with many downstream molecular biology applications. DNA purity and integrity can be determined spectrophotometrically, electrophoretically and with the use of quantitative PCR (qPCR) (Figure 3.7). The application of each of these approaches will be described in the following sections.

3.5.2.1 A260 : A280 Ratio to Evaluate Levels of Protein Contamination The most widely adopted method for determining the purity of DNA is that of spectrophotometry, which involves the evaluation of a number of absorbance ratio metrics. For example, residual protein contamination can be determined by examining the A260 : A280 ratio. This procedure was first described to measure protein purity in the presence of nucleic acids. However, it is now commonly used to assess protein contamination of DNA. It is important to note that the A260 : A280 ratio is an indication of purity rather than a precise measure. Pure DNA preparations have an A260 : A280 ratio of greater than or equal to approximately 1.8. This value can only be approximate due to the different absorption spectra of the bases present in DNA, each of which have a different A260 : A280 ratio (Table 3.3). A number of factors can affect the accuracy of the measurement of the A260 : A280 ratio. For example, readings from dilute samples will have very little difference between the absorbance at 260 and 280 nm leading to inaccurate ratios. In addition, the type(s) of protein present will have an effect. Absorbance in the UV spectrum by proteins is primarily the result of the presence

DNA Extraction from Food Matrices

43

Figure 3.7  An  example decision tree for use in determining DNA purity. Table 3.3  A  260 : A280 ratios for nucleotides. Nucleotide

A260 : A280 ratio

Adenine Cytosine Guanine Thymine

4.50 1.51 1.15 1.47

of aromatic ring structures. Thus, the amino acid sequence of a protein can influence the ability of a protein to absorb UV light. The effects of this can be seen by comparing the extinction coefficients associated with different proteins. For example, bovine serum albumin (BSA) has an extinction coefficient value of 0.7 for a 1 mg mL−1 solution at 280 nm, while streptavidin has an extinction coefficient of 3.4, absorbing nearly five times as much light at 280 nm at the same concentration. A further consideration is that many contaminants strongly absorb light in the UV region of the spectrum and, as a consequence, can affect the ratio calculations. For example, phenol has an absorbance peak at 270 nm and generates an A260 : A280 ratio of 2. Solutions of DNA that are uncontaminated by phenol theoretically have an A260 : A280 ratio of 1.8. In addition to affecting

Chapter 3

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the ratio calculations, contamination by phenol can significantly contribute to overestimation of DNA concentration and potentially inhibit downstream molecular biology procedures. The pH of the solution can also affect the A260 : A280 ratio. Acidic solutions have lower ratios by up to 0.3 and basic solutions have an increased ratio by a similar amount. In practical terms the A260 : A280 ratio should be calculated after correcting for any non-­nucleic-­acid absorbance at A320, using the following formula. DNA Purity

A260  A260  A320   A280  A280  A320 

3.5.2.2 A260 : A230 Ratio to Evaluate Levels of Contamination This ratio is frequently employed as a secondary measure of nucleic acid purity. Strong absorbance values at wavelengths close to 230 nm are indicative of the presence of organic compounds or chaotropic salts in the purified DNA. Substances including urea, EDTA, guanidine HCL and phenolate ions all exhibit absorbance maxima at or close to 230 nm. Therefore, the A260 : A230 ratio can prove useful for evaluating the presence of reagent carryover from the processes involved in the isolation of DNA. As a general guide, the A260 : A230 ratio should be greater than 1.5, and preferably close to 1.8. Values that differ significantly from these target values are usually indicative of extraction reagent carryover.

3.5.3  RNA Contamination Pure RNA has an A260 : A280 ratio of 2.0, therefore if a DNA sample has an A260 : A280 ratio greater than 1.8 it can indicate the presence of RNA contamination. Unfortunately, due to the similarity in absorption profiles of RNA and DNA, the most accurate way of determining RNA contamination is to analyse the sample using agarose gel electrophoresis, where the RNA will be visible as a brightly staining smear migrating ahead of the DNA. RNA contamination is conventionally removed by the addition of DNase-­free RNase A during the DNA isolation procedure.

3.5.4  Non-­nucleic-­acid Contamination Absorbance values at 320 nm can indicate the presence of turbidity in the solution and are another indication of possible contamination.

3.5.5  Evaluation of a DNA Sample's Integrity Agarose gel electrophoresis of isolated DNA complements the data that can be obtained from UV absorbance readings. It can provide an alternative means of determining the concentration of a DNA solution while providing an indication as to the integrity of the extracted DNA.

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45

Figure 3.8  Image  of electrophoresed DNA samples stained with ethidium bromide. Reduced DNA integrity is indicated by the presence of a visible smear, which extends from the main high molecular weight DNA band down the gel. Legend: lane 1 1 kb ladder, lane 2 intact DNA, lane 3 partially degraded DNA, lane 4 moderately degraded DNA, lane 5 highly degraded DNA.

The DNA concentration of a sample can be determined following gel electrophoresis by comparing the staining intensity obtained for a sample of DNA with that obtained for a DNA quantitation standard. In order to visualize the DNA in an agarose gel, staining with a DNA intercalating dye, such as ethidium bromide or SYBR Green, is required (Figure 3.8). The method is useful in situations where the DNA concentration is below that which can be accurately determined using spectrophotometry and in cases where contaminants absorbing at 260 nm make accurate quantitation impossible. However, the most common reason for carrying out a gel-­electrophoretic analysis is to determine the integrity of the isolated DNA. For example, with use of a 1.0–1.5% agarose gel, intact genomic DNA should appear as a compact, high-­molecular-­weight band with no low-­molecular-­weight smears. Degraded DNA frequently appears as a visibly elongated smear with a high proportion of the material positioned towards the bottom of the gel.

3.5.6  Determining the Presence of Inhibitory Compounds A number of methods have been developed to determine if inhibitory  substances are present with DNA that has been extracted from biological samples. A commonly employed approach involves the evaluation of the PCR efficiency generated for a test sample, through the use of a serial dilution of the sample.22 Alternatively, the use of internal amplification controls (IACs) can be adopted, which utilise the addition of an exogenous DNA target into the purified target nucleic acid. The PCR efficiency of a qPCR

Chapter 3

46

Figure 3.9  Diagrammatic  representation of performing a standard curve determination of PCR efficiency.

assay designed to the IAC can then be used to determine the presence of inhibitory compounds (Figure 3.9). The slope of a standard curve can provide an indication of the efficiency of  a real-­time PCR reaction. A slope of −3.32 corresponds to a PCR efficiency of 1, or 100%, and the amount of PCR product doubles during each cycle. Slopes  of less than −3.32 are indicative of PCR efficiencies less than 1, whereas slopes of greater than −3.32 are indicative of PCR efficiencies that appear to be greater than 100%. This can occur when values are measured in the nonlinear phase of the reaction or can indicate the presence of inhibitors. The efficiency of a real-­time PCR assay can be calculated by analysing a template dilution series and plotting the quantitation cycle (Cq) values against the log10 template amount, and determining the slope of the resulting standard curve. From the slope (S), the PCR efficiency can be estimated by applying the formula.  1  PCR efficiency  %   10 S  1   100   PCR efficiency values in the region of 100% ± 15% are generally acceptable, but deviations greater than this are strongly indicative of the presence of inhibition.23

3.6  Concluding Remarks Since the first isolation of DNA by Miescher in 1869,1 a large number of different extraction techniques have been developed which can be applied to a range of different matrices. Any protocol, regardless of whether it uses density centrifugation, organic solvent extraction or DNA isolation kits, is worth

DNA Extraction from Food Matrices

47

considering if it results in good yields of uncontaminated DNA of sufficient quality from a sample. Considering the range of factors which can influence the process of DNA extraction, many laboratories still select the method to employ by the use of a “what works” rule. With some exceptions, molecular biologists still use basic protocols3,24 and incorporate modifications according to their specific requirements. Key modifications include varying the concentration of reagents such as CTAB, NaCl and PVP as well as denaturants (e.g. DTT) and general antioxidant compounds (e.g. ascorbic acid and BME). Although there are examples of methods that have been successfully applied across a range of food matrices,3,24,25 the chemical heterogeneity and diversity of foods and food ingredients makes it difficult for a single method to be used across all possible sample types. Recent technical developments with paramagnetic-­particle-­based systems may help address this problem, while enabling the prospect of automated extraction to be explored.26 Automated nucleic acid extraction procedures offer numerous potential benefits, including reduced working time, decreased labour costs and increased reproducibility and quality of results. However, the development of reliable and portable systems is still required to make this a reality.

Acknowledgements The author gratefully acknowledges funding through the UK Department for Business, Energy and Industrial Strategy (BEIS) as part of the Government Chemist Programme 2017–2020. The author also gratefully acknowledges discussions with fellow colleagues, Malcolm Burns and Gavin Nixon (Foods Team, HS&I, LGC) regarding experiences, expertise and awareness in the field of DNA extraction as applied to food authenticity testing.

References 1. R. Dahm, Discovering DNA: Friedrich Miescher and the early years of nucleic acid research, Hum. Genet., 2008, 122(6), 565–581. 2. R. Dahm, Friedrich Miescher and the discovery of DNA, Dev. Biol., 2005, 278(2), 274–288. 3. M. Murray and W. F. Thompson, Rapid isolation of high molecular weight plant DNA, Nucleic Acids Res., 1980, 8(19), 4321–4326. 4. S. L. Dellaporta, J. Wood and J. B. Hicks, A plant DNA minipreparation: Version II, Plant Mol. Biol. Rep., 1983, 1(4), 19–21. 5. A. K. Lockley and R. G. Bardsley, DNA-­based methods for food authentication, Trends Food Sci. Technol., 2000, 11(2), 67–77. 6. D. G. Peterson, K. S. Boehm and S. M. Stack, Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), a plant containing high levels of polyphenolic compounds, Plant Mol. Biol. Rep., 1997, 15(2), 148–153. 7. N. Shved, et al., Post Mortem DNA Degradation of Human Tissue Experimentally Mummified in Salt, PLoS One, 2014, 9(10), e110753.

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8. N. Gryson, Effect of food processing on plant DNA degradation and PCR-­based GMO analysis: a review, Anal. Bioanal. Chem., 2010, 396, 2003–2022. 9. K. Wilson and J. Walker, Principles and Techniques of Practical Biochemistry, 2000, Cambridge University Press. 10. P. Barnwell, et al., Isolation of DNA from the highly mucilagenous succulent plant shape Sedum telephium, Plant Mol. Biol. Rep., 1998, 16(2), 133. 11. T.-­J. Kang and M.-­S. Yang, Rapid and reliable extraction of genomic DNA from various wild-­t ype and transgenic plants, BMC Biotechnol., 2004, 4(1), 20. 12. N. R. Murphy and R. J. Hellwig, Improved nucleic acid organic extraction through use of a unique gel barrier material, BioTechniques, 1996, 21(5), 934–939. 13. R. N. Pandey, R. P. Adams and L. E. Flournoy, Inhibition of random amplified polymorphic DNAs (RAPDs) by plant polysaccharides, Plant Mol. Biol. Rep., 1996, 14(1), 17–22. 14. A. D. Sharma, P. K. Gill and P. Singh, DNA isolation from dry and fresh samples of polysaccharide-­rich plants, Plant Mol. Biol. Rep., 2002, 20(4), 415. 15. M. E. John, An efficient method for isolation of RNA and DNA from plants containing polyphenolics, Nucleic Acids Res., 1992, 20(9), 2381. 16. D. Puchooa, A simple, rapid and efficient method for the extraction of genomic DNA from lychee (Litchi chinensis Sonn.), Afr. J. Biotechnol., 2004, 3(4), 253–255. 17. U. Pich and I. Schubert, Midiprep method for isolation of DNA from plants with a high content of polyphenolics, Nucleic Acids Res., 1993, 21(14), 3328–3330. 18. J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, 2001, Cold Spring Harbor Laboratory Press. 19. R. Boom, et al., Rapid and simple method for purification of nucleic acids, J. Clin. Microbiol., 1990, 28(3), 495–503. 20. K.-­H. Esser, W. H. Marx and T. Lisowsky, maxXbond: first regeneration system for DNA binding silica matrices, Nat. Methods, 2006, 3(1), 68. 21. T. G. Mayerhöfer and J. Popp, Beer's law -­ why absorbance depends (almost) linearly on concentration, ChemPhysChem, 2019, 20(4), 1439–4235. 22. A. Tichopad, et al., Standardized determination of real-­time PCR efficiency from a single reaction set-­up, Nucleic Acids Res., 2003, 31(20), e122. 23. E. J. Kontanis and F. A. Reed, Evaluation of Real-­Time PCR Amplification Efficiencies to Detect PCR Inhibitors, J. Forensic Sci., 2006, 51(4), 795–804. 24. J. J. Doyle, A rapid DNA isolation procedure for small quantities of fresh leaf tissue, Phytochem. Bull., 1987, 19, 11–15.

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25. S. M. Aljanabi and I. Martinez, Universal and rapid salt-­extraction of high quality genomic DNA for PCR-­based techniques, Nucleic Acids Res., 1997, 25(22), 4692–4693. 26. N. Kovačević, Magnetic Beads Based Nucleic Acid Purification for Molecular Biology Applications, in Sample Preparation Techniques for Soil, Plant, and Animal Samples, ed. M. Micic, Springer, New York, NY, 2016, pp. 53–67.

Chapter 4

“DNA Techniques” Case Study: Isothermal Approaches G. Nixon* LGC Ltd, Teddington, Middlesex, TW11 0LY, UK *E-­mail: [email protected]

4.1  Introduction Modern molecular diagnostics is built upon nucleic acid amplification (NAA) technologies, which have revolutionised the detection and quantitation of biological materials. The polymerase chain reaction (PCR)1,2 was quickly established as the dominant NAA technology due to features such as relative ease of assay design and high levels of assay sensitivity and specificity. PCR-­ based molecular diagnostics, especially quantitative real-­time PCR (qPCR), are integral to current food and feed authenticity testing processes. However, PCR-­based amplification approaches are limited by factors which include the requirement for complex thermal cycling instrumentation, well documented susceptibilities to PCR inhibitors frequently found within biological samples3–6 and an initially complex licensing landscape. Hence, alternative NAA technologies have been developed and commercialised that generally do not require thermal cycling processes (isothermal) and broaden analytical capability. These ‘isothermal’ approaches'7,8 provided the means to circumvent the existing licensing landscape and potentially simplify the nucleic acid amplification process. As well as dispensing with the need for complex thermal cycling instruments, isothermal approaches can also offer advantages, such   Food Chemistry, Function and Analysis No.16 DNA Techniques to Verify Food Authenticity: Applications in Food Fraud Edited by Malcolm Burns, Lucy Foster and Michael Walker © The Royal Society of Chemistry 2020 Published by the Royal Society of Chemistry, www.rsc.org

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“DNA Techniques” Case Study: Isothermal Approaches

51

as rapid reaction time and reduced susceptibility to inhibitors. This has provided the potential to apply molecular methods to applications that were previously difficult to achieve with PCR, such as point-­of-­use (POU) diagnostics. This case study provides a brief overview of isothermal technologies and explores specific applications of isothermal nucleic acid amplification within the food diagnostics sector.

4.2  Main Isothermal NAA Technologies 4.2.1  Cross Priming Amplification (CPA) 4.2.1.1  Overview Cross priming amplification (CPA)9,10 is a relatively new isothermal NAA technology that shows similarities with the LAMP technique (see Section 4.2.3). CPA utilises a strand displacement polymerase (Bst DNA polymerase) with displacement and cross primers (5′ ends not complementary to the template strand) to amplify a DNA template under isothermal conditions within 60 minutes. Multiple primers are involved in the generation of cross priming sites and subsequent amplification of a target sequence at a constant temperature of approximately 63 °C. Original development work by Fang and co-­workers evaluated the performance of a CPA-­based method for Mycobacterium tuberculosis from sputum specimens, which showed an improvement in sensitivity compared with some of the established analysis methods.10 Later developmental work by Feng and co-­workers demonstrated that a CPA-­based test strip (CPA-­ Strip) assay for the rapid detection of mutton from meat mixture11 was capable of specifically detecting the target species at a 1% w/w detection limit.

4.2.1.2  Benefits CPA shares the basic advantages of many isothermal NAA technologies, such as ruggedness and minimal instrumentation requirements. CPA is a rapid and flexible approach that can be applied to real-­time and test-­strip-­based applications.11 Specific CPA-­based methods were found to be 10-­fold more sensitive [2.5 fg limit of detection (LOD)] than the corresponding PCR assay.12

4.2.1.3  Technology Considerations CPA represents an emerging technology without the expansive user base of approaches such as LAMP which may limit development opportunities.

4.2.2  Helicase-­dependent Amplification (HDA) 4.2.2.1  Overview Helicase-­dependent amplification (HDA) utilises the ability of a DNA helicase enzyme to unwind double stranded DNA and expose primer annealing sites for subsequent priming, without the requirement for heat denaturation at a

Chapter 4

52 13,14

single temperature (originally 37 °C, now typically 65 °C). The amplifi­ cation process is based upon a DNA helicase (UvrD helicase) working in association with single-­stranded binding proteins (SSBs) to prevent the separated strands from re-­binding prior to primer annealing. Once hybridisation and extension of the initial primers has occurred, the newly formed DNA duplex is then separated by the helicase and the cycle of amplification continued.13 Thus, a simultaneous chain reaction develops, resulting in exponential amplification of the selected target sequence. An and co-­workers14 developed a modified approach adapted to higher temperatures that was described as thermophilic helicase-­dependent amplification (tHDA) assay and demonstrated increased assay sensitivity (as low as 50 genomic copies).

4.2.2.2  Benefits HDA shares the basic advantages of many isothermal NAA technologies, such as ruggedness and minimal instrumentation requirements. HDA is a relatively simple and rapid isothermal technology which shares features with PCR-­based methods (e.g. use of primer pairs) and is suitable for POU diagnostic applications.

4.2.2.3  Technology Considerations In a similar fashion to CPA, HDA represents an emerging technology without the expansive user base of approaches such as LAMP, which may limit development opportunities. In addition, the assay development process requires careful screening of candidate primer sets to ensure that a useful amplicon is generated from the target sequence.15

4.2.3  Loop-­mediated Isothermal Amplification (LAMP) 4.2.3.1  Overview Loop-­mediated isothermal amplification (LAMP)16 is a relatively well established technology that has seen a large uptake within the research and commercial communities. LAMP amplification is typically conducted at between 60 and 65 °C and exploits the auto-­cycling strand displacement and DNA synthesis activity of the Bst DNA polymerase together with a set of four specially designed inner (FIP, BIP) and outer primers (F3, B3) which collectively target six regions of the DNA sequence (Figure 4.1). The amplification process utilises the forward inner primer (FIP) containing sequences of the sense and antisense strands of the target DNA initiates the LAMP reaction. Strand displacement DNA synthesis primed by the F3 outer primer releases a single-­ stranded DNA product that serves as the template for DNA synthesis primed by a second set of backward inner (BIP) and B3 outer primers, producing a stem–loop DNA structure. Subsequent rounds of LAMP cycling involve an inner primer hybridising to the loop and initiating displacement DNA

“DNA Techniques” Case Study: Isothermal Approaches

53

Figure 4.1  Generation  of LAMP starting materials using the F3, B3, FIP and BIP

primer set. The stem–loop DNA structure forms the basis for the cycling amplification stage which generates complex amplification products.

synthesis that generates the original stem–loop DNA and a new stem-­loop DNA with a stem twice as long. The auto-­cycling reaction proceeds and is capable of synthesising 109 copies of the target in less than an hour.16 A complex set of amplification products is generated that comprises stem–loop DNAs with several inverted repeats of the target and ‘cauliflower-­like’ structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Assay amplification efficiency and sensitivities can be increased through the use of an additional pair of ‘loop’ primers that bind internally.

4.2.3.2  Benefits LAMP shares the basic advantages of many isothermal NAA technologies, such as ruggedness and minimal instrumentation requirements. LAMP is typically found to be more resistant to classical PCR inhibitors found within complex matrices, such as food and clinical samples17 which is likely to be due to differences in DNA polymerase susceptibilities. The increased robustness of this approach means that unprocessed or partly processed samples can be directly analysed.18,19 In addition, the technology has good assay performance characteristics, such as rapidity,20–22 good sensitivity23–25 and excellent specificity (conferred by multiple primer binding)17,26,27 which contribute to overall applicability and usability.

54

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4.2.3.3  Technology Considerations The complex assay design process, which requires four to six target sequences, combined with difficulties in multiplexing due to potential primer–primer interactions within the LAMP primer sets, results in a less flexible amplification technology than PCR. In addition, the complex amplification process generates concatemeric amplification products, which limits the usage of simple detection approaches (e.g. agarose gel electrophoresis) and necessitates the application of fluorophore–quencher approaches.28

4.2.4  Recombinase Polymerase Amplification (RPA) 4.2.4.1  Overview Recombinase polymerase amplification (RPA)29 is a relatively new isothermal technology with a similar mechanism to PCR in which a pair of oligonucleotides bind to the opposite strands of the template DNA and generate complementary sequences using a DNA polymerase. The amplification process is based upon three core components – a recombinase enzyme, DNA binding proteins and a polymerase enzyme. Unlike the PCR process, RPA utilises recombinase–primer complexes to identify and initiate strand displacement, which removes the need for thermal template denaturation. The T4 uvsX recombinase strand displacement activity is stabilised by single-­stranded DNA binding proteins (T4 gp32) and primer extension is catalysed by the strand-­displacing DNA polymerase Pol I (Bsu). Exponential amplification is achieved by the cyclic repetition of this process, which is typically completed within 30 minutes.29,30

4.2.4.2  Benefits RPA shares the basic advantages of many isothermal NAA technologies, including ruggedness and minimal instrumentation requirements. RPA technology is characterised by rapid amplification times (reaction typically completed within 30 minutes) and good sensitivity (single copy level)29 which lends itself to diagnostic applications, such as point of use testing.31,32 RPA is a low temperature (usually between 37 and 42 °C) isothermal technology that only requires basic heating equipment, which minimises instrumentation costs and broadens the testing environment. Additional functionality includes the availability of a real-­time fluorescent probe chemistry and the relative ease of multiplexing.29,33

4.2.4.3  Technology Considerations In comparison to PCR, the assay design guidelines are less well developed, which means that a more complex design and evaluation process is required in order to generate suitable designs.

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55

4.3  PCR vs. Isothermal Technologies PCR-­based tests represent the ‘gold standard’ approach within the molecular diagnostics community due to their well characterised performance features and the broad range of compatible and cost-­effective reagent and instrument systems available throughout the testing community. The latest generation of isothermal NAA technologies offer a compelling set of features with the potential to outperform routine PCR methodologies. Table 4.1 compares the core performance characteristics of PCR against a limited selection of isothermal NAA technologies and highlights the main areas of divergence, such as reaction times and thermal profiles. A well reported advantage of many isothermal techniques relates to the rapid amplification times (typically less than 30 minutes) delivered using simple heating instrumentation as compared with PCR using thermal cyclers. However, it should be noted that modern rapid thermal cyclers in combination with fast PCR chemistries and appropriate assays are capable of performing PCR amplifications in less than 30 minutes.34,35 Achieving these PCR performance improvements requires complex thermal cyclers, which result in additional cost and places PCR at a disadvantage to a standard isothermal setup. Another central difference between PCR and isothermal-­based approaches relates to the reported tolerance of isothermal chemistries to common matrix interferents.36,37 This is primarily due to use of alternative DNA polymerases to Taq DNA polymerase which exhibit different inhibitory profiles and means that direct or minimal sample processing processes can be employed with isothermal approaches.

4.4  Food Applications Isothermal NAA technologies offer a wide range of potential benefits to the food testing community, such as improving the availability and uptake of point-­of-­use applications and reducing the effects of complex food matrices upon diagnostic performance. These technologies have the potential to support food legislative frameworks within the UK and/or European Union (EU), such as food labelling regulations.38 The following applications highlight the scope of these technologies within the food testing sector.

4.4.1  Meat Speciation The fraudulent substitution of meat represents a serious issue of economic, health and social significance. Meat speciation testing is a relatively well developed area for isothermal NAA-­based diagnostics with demonstrated applications ranging from the detection of common adulterants, such as pork and chicken, to exotic meats.11,19,39,40 Sul and co-­workers19 recently developed a LAMP-­based methodology targeting the 16S rRNA gene for the

Table 4.1  Table  of selected isothermal NAA technologies compared with PCR.E denotes endpoint detection and RT denotes real-­time detection options. Feature

Gold standard

Name

Polymerase chain reaction (PCR) 50–95 °C (Thermal cycling) Simple Yes Electrophoresis (E) Fluorescence (RT)

Cross priming Helicase-­dependent Loop-­mediated isothermal Recombinase polymerase amplification (CPA) amplification (HDA) amplification (LAMP) amplification (RPA) 60 °C 37 °C or 60–65 °C 60–65 °C 37 °C Simple Yes Electrophoresis (E) Fluorescence (RT)

Simple Yes Electrophoresis (E) Fluorescence (RT)

Denaturing Agents

Thermal

Helicase

Typical detection   time Key References