Detection, identification and quantification of GMOs and derived products: present and future challe

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Detection, identification and quantification of
GMOs and derived products present and future
challenges
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Overview

• Who needs detection methods, and why?
• Detection methods and ENGL
• Requirements for GM-detection
• Target molecules for GM detection
• GM quantification
• Limit of detection (LOD) and quantification
  (LOQ)
• The QPCRGMOFOOD project

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Who needs detection methods,and why?

• GMO producers
• To assure purity and segregation of products
• To be able to trace genetic modification in
  breeding
• Food feed industry, seed companies
• To assure purity and segregation of products
• To assure compliance with legislation
• Competent (enforcement) authorities
• Product control, compliance with legislation
• To be able to retrieve specific products
• E.g. if marketing permission withdrawn
• Laboratories
• To provide services to society (incl. above)

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Detection methods and ENGL

• ENGL Network for competent authorities
• Focus on control and legislation
• Currently DNA based methods
• (Protein based methods primarily for industry)
• Focus on challenges and possibilities
• Contribute to improving detectability
• Develop and improve control schemes and methods
• ENGL initiated EoI for NoE FP6 EUNATYM-GMO
• Communicate results to society (incl. industry)
• Give advice to society

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Requirements for GM-detection

• Target molecules must be present
• DNA (template) or protein
• Capture molecules must be developed
• For DNA primers and probes
• For proteins antibodies
• Reference materials
• Positive and negative controls
• Calibrants for quantitation

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Requirements for GM-detection .and corresponding
challenges

• Target molecules must be present
• Degraded or absent target molecules undetectable!
• Effects of processing usually significant
• Important to improve methods to recover target
• Capture molecules must be developed
• Impossible without (description of) target
  molecules
• Need material for method development / validation
• Reference materials
• Material for production of RMs difficult to
  obtain
• Materials must serve many different functions

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Requirements for GM-detection .and two
particular challenges

• Gene stacking
• Hybrids between authorised GMOs
• Detection methods will detect GMO1 GMO2
• In Europe, separate authorisation required (not
  in USA)

• Unknown GMOs
• Not described in any available documents
• Not subject to normal safety evaluation
  procedures
• ENGL initiated EoI FP6 Measure-GMO

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GM target sequences categories and
applications

• GMO - defined by specific transformation event
• In official documents and notifications
• Transformation of plants usually insertion of
  DNA
• Composition of inserted DNA and insertion locus
• Four levels of specificity of GM target sequence
• Screening ? event specific identification

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Transformation of plants

• Usually insertion of DNA
• Could also be deletion or substitution
• Insertion only focus hereafter
• DNA inserted the gene construct
• Composed of several elements
• Some elements natural, some synthetic
• Some elements widely used, other unique
• Number of inserted copies may vary
• And/or additional fractions of gene construct

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Gene construct
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Levels of specificity GM targets (DNA)
Screening targets
Gene specific targets
Construct specific
Event specific targets
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Field of application

• Screening targets
• May yield false positives and false negatives!
• Not suited for identification and quantification
• Cost efficiency the main motivating factor
• Gene and construct specific targets
• Positives ? GMO presence
• Event specific targets
• Well suited for identification and quantification

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Reference genes (for plants)

• A GMO belongs to a species
• Reference gene
• Demonstrates presence of DNA from species
• Quantification of species DNA
• Reference for GMO quantification

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PCR based quantification

• At least four different types of assays
• Competitive PCR
• Real-time PCR
• Arrays
• Capillary electrophoresis
• Determine initial copy numbers of targets
• Reference (species) and GM target
• Comparison of relative quantities
• Reference target to GM target

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Limit of detection (LOD) and quantification
(LOQ)

• Lowest amount reliably detected or quantified
• Usually 95 confidence
• Absolute limits copy number based
• Relative limits ratio or percentage based
• Practical limits relative limits applicable for
  sample

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Limit of detection (LOD) and quantification
(LOQ)

• The absolute LOD is the lowest number of GM
  target copies (e.g. 10) that will be detected 95
  out of 100 times. This is method specific
• The maximum number of reference target copies
  (e.g. 1 million) that can be included in the test
  determines the relative LOD. This is also method
  specific
• E.g.

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Limit of detection (LOD) and quantification
(LOQ)

• If the number of observed reference gene copies
  is 2000 in the test, then with the above example
  the practical LOD 0.5. This is sample
  specific, because

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Limit of detection (LOD) and quantification
(LOQ)

• Legal threshold for labelling in EU 1 percent
• Test reports therefore refer to relative LOD/LOQ
  ()
• Relative LOD/LOQ usually determined for method
• Under exceptionally favourable conditions
• Therefore if reported as sample specific
  unrealistic!
• Practical LOD/LOQ must also be determined
• Depending on absolute LOD/LOQ and template DNA
• Usually determined by quantitation of reference
  gene

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Qpcrgmofood QLK1-1999-01301

• Reliable, standardised, specific, quantitative
  detection of genetically modified food
• Initiated through CEN on basis of identified
  needs
• Six workpackages
• DNA extraction methods
• Reference genes for plant species
• Characterisation of event specific sequence
  motifs
• GM detection / quantification method development
• Method validation
• Socio-economic studies, relation to traceability
  methods

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Reference genes for plant species

• Basis for quantitation
• Identify presence of DNA from the species
• Before testing for specific GMOs
• Wide range of cultivars of the species tested
• Copy number and DNA sequence stable
• Several candidates rejected as a result of
  specific tests
• Non-target species also used to test specificity
• Output Suitable primers and probes for real-time
  PCR
• Now ? validation? proposed as standards to
  CEN/ISO

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Event specific sequences

• Required for development of event specific
  methods
• Provides better insight in actual process of
  transformation
• Useful to verify and update genetic maps of GMOs
• Provides information of relevance to assess
  stability of the genetic modification
• Relevant to risk assessment

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GM detection/quantification method development

• Results so far
• Soybean
• Methods for one GMO
• Maize
• Methods for seven GMOs
• Rapeseed
• Methods for three GMOs
• Additional GMOs still in progress

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Take home messages

• Major challenges
• Access to reference materials of GMOs
• Better DNA extraction protocols
• Cost reduction, multiplexing
• Expression of LOD/LOQ on certificates
• Unauthorised GMO (those that we dont know)
• Major achievements
• Identification of reliable reference genes
• Characterisation of transformation events
• Development of event specific quantitative PCR
• Multiplex methods and determination of LOD/LOQ

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Take home messages

• Major challenges
• We believe we know what they are
• Collate resources and do the job
• Major achievements
• We believe we can cope with the challenges and
  find good solutions
• ENGL and associated EoIs

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