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Detection and Identification Online Discussions 2019

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Detection and Identification Online Discussions 2019 [#9976]
Distinguished Colleagues,

The Detection and Identification Online Discussions 2019 provides an opportunity to update and share our experiences on current trends in the detection and identification of LMOs and emerging issues in the detection, identification and monitoring of organisms, components and products of synthetic biology. Also open for discussions are the current and existing capacities for the detection and identification of LMOs.
The feedback on these issues will be summarized as a resource for the network of laboratories for the detection and identification of LMOs.
We still have some time and therefore encourage meaningful engagements among participants. The Secretariat echoes the moderator’s invitation to participants to contribute to the discussions while thanking those that have done so already..

Best regards,
Shakirat Ajenifujah-Solebo
posted on 2019-11-01 08:24 UTC by Ms. Shakirat Ajenifujah-Solebo, Nigeria
RE: Detection and Identification sources of methods [#10034]
Dear participants,

A number of peer-reviewed sources of information are available in addition to the ENGL handbook.  Unlike regional handbooks, these resources discuss detection methods in the global context.  Several are open access.  For example, a discussion on sampling is available (Freese et al 2015) and a comparison of DNA and Protein-based detection methods (Alarcon, 2019) will be open access as of January 2020. Another good source of information is the book “Immunoassays in Agricultural Biotechnology“ edited by Guomin Shan (2010), which discusses the way that protein-based tests can be applied across the food chain.

Additionally the International Seed testing Association maintains a site with a list of sources of testing information at https://www.seedtest.org/en/analytical-methods-_content---1--1477.html and links from this page. 

In response to the post of Apichart Vannavichit, [#10016] and Dr. Sarah Agapito-Tenfen [#10021], among others, the work of International Standards Organisation Technical Committee 34 subcommittee 16 (TC34/SC16) on detection methods is notable.  A number of ISO standards have been published that cover PCR and protein-based analytical methods, and work is in progress on a sampling standard.  The most relevant are listed below.

Guomin Shan (Ed.) 2010.“Immunoassays in Agricultural Biotechnology”, John Wiley & Sons, Inc.   ISBN 978-0-470-28952-5

Clara M. Alarcon 2019. Application of DNA- and Protein-Based Detection Methods in Agricultural Biotechnology.  J Agric Food Chem. v67:1019-1028. doi: 10.1021/acs.jafc.8b05157.

Freese, L., Chen, J., and Shillito, R. Sampling of Grain and Seed to Estimate the Adventitious Presence of Biotechnology-Derived Seeds in a Lot. Cereal Foods World, v60, pp. 9-15, 2015.  DOI http://dx.doi.org/10.1094/CFW-60-1-0009

Relevant ISO TC34/SC16 standards – further information can be found at:  https://www.iso.org/committee/560239.html
ISO 16577: Molecular biomarker analysis -- Terms and definitions
ISO 24276: Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- General requirements and definitions
ISO 21569: Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- Qualitative nucleic acid based methods
ISO 21570: Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- Quantitative nucleic acid based methods
ISO 21571: Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- Nucleic acid extraction
ISO 21572: Foodstuffs -- Molecular biomarker analysis -- Protein-based methods
ISO/ TS 16393 Molecular biomarker analysis -- Determination of the performance characteristics of qualitative measurement methods and validation of methods
ISO 16578: Molecular biomarker analysis -- General definitions and requirements for microarray detection of specific nucleic acid sequences

The following standards are under development:
ISO/CD 20813: Molecular biomarker analysis -- Methods of analysis for the identification and the detection of animal species from foods and food products (Nucleic acid based methods) -- General requirements and definitions
ISO/AWI 22753: Molecular biomarker analysis -- Methods of analysis for the detection of genetically modified organisms -- Semi-quantitative method for the statistical evaluation of weight/weight GMO content in seeds and grains
ISO/NP 22942-1: Molecular biomarker analysis -- Nucleic acid isothermal nucleic acid amplification based methods -- Part 1: General requirements
posted on 2019-11-08 02:36 UTC by Dr Raymond Shillito, BASF Corporation
RE: Detection and Identification of products of gene editing [#10036]
Dear forum participants,

A requirement to detect and uniquely identify an organism developed using biotechnological tools may be connected to its regulatory status. Where the organism is regulated as an LMO/GMO, developers may be required to develop methods that enable the organism to be detected in trade once it is commercialised, e.g. in bulk shipments of grain.
However, in the context of the protocol, these methods are only relevant for LMOs are likely to cause adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health. 

Methods used in the regulatory field are often validated PCR-based methods, although protein-based methods are also a valid and much more economical approach. Methods must also conform with certain performance criteria as set out in the Codex guideline CAC/GL 74-2010, and with regulatory requirements, and be practically feasible for use with bulk commodities. Such PCR and protein-based methods and links to Certified Reference Material providers are routinely posted in the database: http://www.detection-methods.com. Where organisms are not within the scope of LMO/GMO regulation in a country or trading block, there should not be a requirement for a specific detection method to uniquely identify them, as expressed by Apichart Vannavichit, #10016. For crops developed using genome editing, there are currently a range of approaches to their regulation, with certain applications regulated as LMOs/GMOs and therefore detection methods may or may not be required. For such organisms, the ability to develop sufficiently sensitive detection methods depends on the type of edit. Where the edit involves the incorporation of a DNA sequence of sufficient size for the design of PCR primers (e.g. a gene cassette), PCR-based detection methods can be developed – this is same approach used for the development of detection methods for currently commercialized LM/GM crops.

The development of detection methods is less straightforward where applications of genome editing in plants have comparable outcomes to conventional breeding approaches. While it may be technically feasible to detect small DNA sequence changes in single plants and seeds, detection in bulk seed and grain samples will be much more difficult, even impossible, despite claims to the contrary.  The currently used methods are likely to be stretched to their technical limits, prone to errors and misinterpretation due to false positives, and they are unlikely to meet the established performance criteria applied to LM/GM crop detection methods. The use of screening methods commonly used by a number of participants in this forum to detect LM/GM crops (e.g. #9974 #9990, #9992, #9997, #10012) will not be practical, as each edit is unique, thus leading to a high cost of detecting and diversion of precious resources that could be used for more important societal issues to try to detect an essentially safe product through the application of 10’s and eventually hundreds of separate assays.

We support the position of Pr Stephen Ghogomu, Cameroon in post #9981 in Topic 2, regarding the inability of current analytical methods to “distinguish between the natural and the synthetic/chemical counterparts”.  While it may be technically feasible to detect small DNA sequence changes in single plants and seeds, it is not possible to distinguish or determine how a specific change arose during the breeding process – as a result of the use of a genome editing tool, conventional mutagenesis, or spontaneous mutations – because their outcomes are similar or the same.  This was also the conclusion of the European Network of GMO Laboratories (ENGL, 2019) report, and it adds a further complication to the detection of organisms developed using certain applications of genome editing in bulk shipments.

Claims by Bertheau (2019) to be able to distinguish targeted single base changes from those that occur spontaneously in plants due to claims that ‘plant genomes are stable’ are inaccurate.  The plasticity of plant genomes is well-documented in the scientific literature, with point mutations, insertions, and rearrangements commonplace (e.g. Arber 2010; Custers et al 2019; Schnell et al 2015; Weber et al 2012).  Moreover, the Central Committee on Biological Safety (ZKBS), a voluntary expert panel responsible for evaluating GMOs sponsored by the German Federal Office of Consumer Protection and Food Safety, has rebutted these claims stating: “The option suggested by Bertheau to retroactively identify an edited base within a plant genome and the technique used to generate it is non-existent. The proposed methods are not based on current scientific knowledge and furthermore involve highly variable biological parameters (like epigenetic changes) that are no reliable base for identification.” The ZKBS also referred to the conclusion of the ENGL report: “the report of the Joint Research Centre (JRC) of the European Commission and the European Network of GMO Laboratories (ENGL) that the hints from characterization of a plant that point towards genome editing as quoted by Bertheau are not sufficient to identify which technique was initially applied.”

References cited:

-- Arber W (2010) Genetic Engineering Compared to Natural Genetic Variations, New Biotechnology 27: 517-521. 

-- Bertheau Y (2019). In: Encyclopedia of food chemistry (p. 320-336). DOI: 10.1016/B978-0-08-100596-5.21834-9

-- CAC/GL 74-2010 Guidelines on performance criteria and validation of methods for detection, identification and quantification of specific DNA sequences and specific proteins in foods. Available at: http://www.fao.org/fileadmin/user_upload/gmfp/resources/CXG_074e.pdf.

-- Custers R, Casacuberta JM, Eriksson D, Sági L, Schiemann J (2019) Genetic alterations that do or do not occur naturally; consequences for genome edited organisms in the context of regulatory oversight, Frontiers in Bioengineering and Biotechnology 6: 213. doi:10.3389/fbioe.2018.00213.

-- European Network of GMO Laboratories (ENGL) report endorsed by the ENGL Steering Committee, 26 March 2019:  “Detection of food and feed plant products obtained by new mutagenesis techniques”.

-- On the Identifiability of Genome Editing in plants, September 2019 – ZKBS-commentary on Y. Bertheau (2019), http://www.zkbs-online.de/ZKBS/EN/01_Aktuelles/ZKBS-commentary%20on%20Y.%20Bertheau%20(2019)/ZKBS-commentary%20on%20Y.%20Bertheau%20(2019)_basepage.html viewed on November 6, 2019.

-- Schnell J, M Steele, J Bean, M Neuspiel, C Girard, N Dormann, C Pearson, A Savoie, L Bourbonnière, P Macdonald (2015) A comparative analysis of insertional effects in genetically engineered plants:  consideration for pre-market assessments, Transgenic Research 24: 1-17.

-- Weber N, C Halpin, LC Hannah, JM Jez, J Kough, W Parrott (2012) Crop genome plasticity and its relevance to food and feed safety of genetically engineered breeding stacks, Plant Physiology 160: 1842-1853.
posted on 2019-11-08 02:50 UTC by Dr Raymond Shillito, BASF Corporation
RE: Applicability of Screening Methods [#10037]
Dear forum participants,
the applicability of current techniques for the detection and identification of LMOs becomes more complex as more LMOs are developed and contribute to agriculture.

The posts of Emmanuel #9974 (Costa Rica) and Stephen #9980 (Cameroon) bring up good questions about the use of screening methods for the presence of LMOs.  While early events used Cauliflower (and other) mosaic virus and Agrobacterium sequences, more recently the use of these sequences in products released from major trait provider companies has been reduced.  However, screening methods remain in wide use due to their relative efficiency for absence testing.

We are glad to see that Emmanuel in post #9974 is taking care to check for the possible presence of viral and bacterial sequences that are common in nature are not causing false positive results.  This is important and appears to be applied only rarely.
Cauliflower mosaic virus is very common in brassicas and low levels can lead to results that are interpreted as low levels of the presence of LMOs even where the levels are so low as to represent less than one seed – clearly not indicative of an LMO seed being present in the sample.  This is especially problematic in complex mixtures such as soups where brassicas may be used as thickening agents.   Indeed, there is limited literature on this topic.  Cankar et al. J. AOAC International, V88, pp. 814-822 2005 is one article that does address this issue.

If there is interest in standardization of such methods, it is possible that a Technical Specification could be developed though ISO (this would be in the scope of ISO/TC 34/SC16).
posted on 2019-11-08 02:57 UTC by Dr Raymond Shillito, BASF Corporation
RE: New techniques for Detection and Identification of LMOs [#10038]
Dear forum participants,
new detection methodologies such as digital PCR often promise to be more sensitive, reliable and accurate.  If methods are available the question is then ‚are they applicable‘, in terms of being able to be used locally.
Many of these new and existing techniques are costly both in terms of equipment and supplies.  This is an issue that has been expressed by by Mr. Wendmnew Abrie Mekonnen, Ethiopia in post #9989. The cost of operating such new technologies must be assessed against the possible presence of the LMOs that may be detected, and whether such LMOs are likely to cause adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health.  Each country must address applicability of new techniques – that is whether to invest in new technologies, to use simpler and robust older methods that are still able to provide the desired results, or to direct those resources to more important societal issues.  

It is not necessary to purchase kits for all of the components used in PCR.  The basic solutions can be prepared (much as we did in most of the world (and my lab) in the 1990’s) and the enzymes and nucleotides purchased separately.  This may be a much more effective approach.

In addition, the Protocol does not require that a signatory have a laboratory and test for the presence of LMO’s – just that they have biosafety policies in place.  Regional commercial laboratories may be the most effective way to effect such policies without national governments needing to invest in large infrastructure to test a relatively low number of samples.

Next Generation Sequencing (NGS) has been suggested as an option for detection of products developed using genome editing by Mrs Judith Okolo ( #10017) ,and M Sc. Eva Patricia Bermúdez Garcia (#10012) and is being discussed in the global analytical community.  While such a technology may be useful to detect a known event in a single plant or seed, its applicability to bulk samples such as shipments of grains is doubtful for two reasons.  The first is sensitivity, in that the plant genome is very large (compared to bacteria where such screening methods have been employed, and the second is that like other methods it cannot differentiate between DNA sequence changes arising from spontaneous mutations or other breeding processes, and those caused by genome editing.

On the other hand, new techniques that are being developed for use for the detection and identification of LMOs using isothermal DNA technologies promise to be easier and less expensive than real-time or digital PCR to deploy.  There are 6 or more different approaches to isothermal DNA amplification, and some are more robust than others. These have yet to be fully proven.  International standardization of the principles of this isothermal methodology is under way through ISO/TC 34/SC16.  Such methods may be useful for the detection of LM seeds; it is unlikely though that they will be applicable to products developed using genome editing with single base pair or small DNA changes in bulk samples.
posted on 2019-11-08 03:02 UTC by Dr Raymond Shillito, BASF Corporation
RE: New techniques for Detection and Identification of LMOs [#10066]
Dear Raymond, your contributions have been enlightening. Thank you for the many links to useful resources that you shared.

Interesting point about the cost of using new technologies vis a vis the effects of such LMOs on conservation,  biodiversity and human health, particularly when existing (older) techniques will provide the desired result. And the insight into the possibilities of isothermal DNA technologies for LM seeds.

Kind regards

posted on 2019-11-09 23:01 UTC by Ms. Shakirat Ajenifujah-Solebo, Nigeria