Select Committee on European Communities Second Report - Written Evidence

Memorandum by LGC Limited (formerly the Laboratory of the Government Chemist)


  1. DNA methods based on PCR amplification of specific DNA markers of genetic modification have been successfully developed and applied to the identification of a variety of GM foods including processed products derived from GM soya, GM maize, GM tomatoes and GM potatoes.

  2. Some, more highly processed, foodstuffs may, however, contain very degraded DNA and/or contain PCR inhibitors, both of which factors may affect the PCR reaction such that there may be substantially decreased assay efficiency, or even no reaction at all. This could result in the reporting of false negative results for GM foodstuffs if appropriate controls and reference standards are not employed. To a certain extent these effects may be overcome by modification of the DNA extraction process and PCR assay design and conditions, at additional analytical time and cost. However, if the GM DNA is removed from the product as with some refined foodstuffs or totally degraded, detection is not possible even if the foodstuff originates entirely from a GM crop.

  3. Lower cost "routine" GM food screening options are more reliably applied to raw or moderately processed foodstuffs or ingredients. Typically, batch analysis of these type of foods is offered by analytical laboratories for approximately £100/sample (+VAT). Considerable extra costs are incurred for the analysis of more "complex" samples, generally determined on a food by food basis.

  4. Biotechnology developments, including antibiotic resistance gene removal, and the development of alternative GM control elements, coupled with the ongoing drive to market of new GM crops and foods will necessitate further research and development of new GM tests to ensure the continued validity of the PCR testing process.

  5. The introduction of a "de minimis" threshold for GM material in foodstuffs will necessitate the development of validated quantitative PCR assays; at present these could only be considered semi quantitative. The design of suitable PCR quantitative assays for GM foodstuffs which will have the accuracy and precision to be applied for enforcement of labelling regulations, will post significant technical challenges, and would require Government funding.

  6. In conclusion, the significant challenges to GM food detection by PCR, posed by processed foodstuffs, are such that the food labelling statement "does not contain" in reality should actually state in many cases "cannot be detected and/or quantified using currently available technology".


What are genetically modified (GM) foods?

  7. "GM foods" originate from organisms, generally plants, which have had their DNA altered, or "foreign" DNA introduced by the process of genetic engineering usually for the purpose of:

    —  Enhanced product quality

    —  Increased pest resistance

    —  Introduced agronomic trait

  8. The "foreign" DNA introduced during the genetic engineering process (Figure 1) can act as a "tag" or marker for genetically modified (GM) plants. Detection of these "foreign" genetic markers in food can therefore be used as the basis for development of tests for GM foods.

Plant genetic engineering

    Figure 1. Illustration of a method of production of a genetically modified crop plant by infection with transformed agrobacterium (adapted from Biotechnology for Crop Improvement p7 in Recent Advances in Plant and Microbial Biotechnology, AFRC/NCBE).

  9. In a typical genetically modified organism there are usually several DNA markers that may be used for detection:

    —  the gene(s) introduced into the organism to elicit the required new characteristic; e.g., the gene introduced into Monsanto's Roundup Ready soya to make the crop resistant to glyphosate herbicide.

    —  the "start" (promotes) and "stop" (terminator) DNA sequences, which flank the introduced gene and act as "molecular switches" to ensure that the introduced gene functions properly;

    —  chemical resistance markets, e.g., antibiotic resistance, which are introduced into the plant to aid selection and development of the GM plant.

GM crops approved for field release and food use

  10. Table 1 illustrates the great variety of genetically modified plants grown for food production which have received regulatory approval for field release by various competent authorities worldwide (predominantly in the US). Those marked with an asterisk have received EU approval. It is likely that most of these will also be approved for food use—several already have been, as indicated in the table. GM crops depicted in bold type may be considered as commodity crops which are likely to be mixed with unmodified crops, and/or partially processed in the country of production, as is currently the case the GM soybeans and maize.



  11. These foods can be readily identified as GM in their raw state (see Detection), but most of them are likely to be processed to varying degrees and mixed as ingredients in complex foodstuffs which pose more challenging analytical problems (see Limitation).

  12. As identified in table 1, there are several crops now approved for food use within the EU. GM soybean and maize have resulted in the greatest level of public debate. Issues with respect to detection of these GM commodity crops in foodstuffs are discussed below.


Methods for detection of GM foods

  13. Various methods have been developed for the detection of genetically modified organisms. Labelling regulations state that GM protein and/or DNA based methods may be used for analysis.

Protein based methods

  14. Protein based methods (immunological and enzymic) detect the gene product or metabolites whose production is influenced by the gene product. These methods have two significant limitations so are not routinely employed by most laboratories for GM food detection. Antibodies have to be raised to the specific proteins produced as a result of the genetic modification, and very few relevant antibodies are available. More critically, proteins degrade on processing, so even if antibodies exist, the methods are generally only applicable to fresh, raw foodstuffs.

DNA based methods

  15. DNA based methods are the most reliable for the identification of genetic modifications, and have been most widely used. At a practical level, DNA based testing for GM food involves first extracting DNA from the food sample. Some foodstuffs such as tomato puree are more challenging, and it may be necessary to try several extraction and purification procedures in order to extract DNA suitable for analysis. Genetic markers indicative of the genetic modification (as described above) are then detected using a very specific and sensitive DNA amplification and detection technique called the polymerase chain reaction (PCR) (see appendix 1). DNA sequences called primers can be synthesised in the laboratory that are designed to bind specifically to the genetically modified DNA if extracted from a food sample. The PCR reaction copies, or amplifies, the DNA designated by the primers, which is then typically identified by simple gel analysis and visual detection of specific bands as illustrated in Figure 2.

Figure 2 GM soya detection by PCR analysis using primers specific for GM "Roundup Ready" soya. Lanes 2-4 nonGM soya samples, lanes 5-8 GM flour samples, lane 9 GM soyabean, lane 10 oilseed rape seeds

  16. DNA is a remarkably stable molecule and often survives food manufacturing processes. Tests can therefore be carried out not only on raw foods but on cooked and processed products.

  17. An ideal PCR screening method is designed such that:

    —  primers selected are specific for genetic elements in a number of GM crops (see Appendix 2 for a table of typical genetic markers used in PCR analysis);

    —  the genetic element/marker should not occur naturally in the plant or in likely contaminating micro-organism;

    —  only a small DNA fragment needs to be PCR amplified to allow application to processed samples in which the DNA is likely to be very fragmented.

  18. At present, using a limited number of PCR reactions the majority of commercially released GM crops/foodstuffs may be identified (see appendix 3). This generic approach to GM food detection, employing the most commonly used markers for plant genetic modification, has significant advantages in that a specific assay does not have to be individually designed for every single GM crop.

  19. However, in order for a GM food testing regime to remain valid, GM targets selected would have to be continually updated to include new/changed "second generation" marker sequences.

  20. It is likely that, in future, plant regulatory sequences will be used to control expression of introduced DNA. Similarly pressure on companies to replace/delete genes conferring antibiotic resistance and diversification of transcription terminators will reduce the utility of widely employed markers.

  21. Experienced analytical laboratories will be able to continually match the new generation of markers with new PCR tests for GM food identification. However, it is likely that funding will need to be provided by Government for the ongoing R&D necessary to maintain the validity of such PCR based tests in support of GM food labelling regulations. Furthermore, this will increasingly necessitate extended knowledge of the genetic modification, particularly as more organisms are brought into the food chain, e.g., GM fish, GM micro-organisms. Commercial sensitivities may also limit access to the required information, a potential constraint which may have to be overcome through regulatory action.

Quality control—ensuring valid experimental results

  22. When carrying out tests on genetically modified organisms it is important to minimise the potential for incorrect analysis. This can be achieved by carrying out appropriate imitation control PCR reactions, using DNA primers designed to recognise whether any plant DNA is present and/or the DNA from the specific crop, e.g., soya or maize is present in addition to the specific genetic modification of interest. These additional PCR tests help guard against false negative results (as may occur through PCR inhibition, see below) and increase confidence in the analysis by confirming the presence of DNA from these crops in the extracted sample.

PCR application to a variety of foodstuffs

  23. At LGC we have successfully applied PCR to the analysis of a wide variety of foodstuffs:
Examples of processed foods from which DNA has been extracted and PCR amplified at LGC

Soya protein isolatesTomato soup Tinned fish1
Soya gritsTomato puree1 Pate
LecithinTomato ketchup Processed meat products
BeanfeastSundried tomato Canned meat products1
Maize starch1Chips Petfood
Maize glutenPotato salad Animal feeds
Pasta and noodlesSmash Biscuits1
Flour samples (maize and soya)Oxo cubes Confectionary bars1
Powdered soups

1 Indicates sample types where DNA extraction and PCR are highly variable, and success cannot be guaranteed.

  24. As indicated with 1 in the above table many processed foodstuffs give highly variable results with PCR. The potential reasons for this are discussed below.

  25. DNA detection methods are not applicable if, in the course of the food production and/or processing, plant DNA is completely separated or destroyed e.g., refined sugars or oils. However, again for these types of food product, the extent of processing may give rise to variability in GM detection. For example, DNA may be able to be extracted from raw pressed oils but not the heavily refined oils, and LGC's practical experience with maize starch has indicated that GM maize could be identified in samples from some sources but not others, presumably due to differences in processing.


  26. Although PCR is a potentially powerful detection assay for the rapid, sensitive and specific identification of GM foods, food processing can significantly influence the validity of the PCR assay.

DNA degradation

  27. Various factors contribute to the degradation of DNA in processed foodstuffs: chemical, physical and enzymatic e.g.

    —  prolonged heat treatment such as autoclaving used in the canning process, may result in DNA hydrolysis which fragments the DNA, or modifies the chemistry of the DNA in such a way that the PCR process may not work. For this reason canned products can give inconsistent results.

    —  increased chemical modification and hydrolysis of DNA at low pH (e.g., vinegar). For example, these factors make tomato puree (and related tomato products such as ketchup) extremely difficult to work with; results are inconsistent and we, in common with many other workers, often fail to achieve successful amplification even with primers designed to detect any plant DNA.

    —  enzymatic degradation of DNA by nucleases may also occur on prolonged storage of fresh foodstuffs.

PCR inhibition

  28. A recognised problem in using PCR methods with foods is the presence of PCR inhibitors that reduce the efficiency of the genetic amplification process. These include many common food components:

    —  cations e.g., Ca2+, Fe3+;

    —  trace heavy metals;

    —  carbohydrates;

    —  tannins, phenolics;

    —  salts e.g., NaC1, nitrites.

  29. Work at LGC, in addition to studies reported in the literature, indicates that the degree of PCR inhibition is to a great extent dependent on the food type, e.g., boiled ham shows little or no inhibition, whereas various kinds of soft cheese completely inhibit the reaction. This could lead to potential analytical problems if for example three pies with fillings of ham, soft cheese, soft cheese and ham were analysed for GM soya content.

  30. Without the proper reference standards and PCR controls, as outlined above in section 2.2, PCR inhibition may easily lead to false negative results, particularly if the GM food analysis is being undertaken by laboratories with insufficient experience with food analysis by PCR to recognise the problem.

  31. It is sometimes possible to overcome these inhibitory effects by extensive dilution of the DNA extract, however, this may not be an option when the amount of DNA in the sample is limiting. In these cases, further purification of the DNA, or the addition to the reaction of PCR enhancers may reduce the level of inhibition. Knowledge of likely inhibitory components in foodstuffs can inform the type of analysis undertaken and modifications to the routine extraction and PCR procedure. Such special analytical modifications may prevent false negatives and allow a higher level of confidence in the result obtained, but add time and cost to the analysis.

  32. It should be noted that if a foodstuff labelled "does not contain [GM material]" becomes subject to forensic analysis as a result of trading standards enforcement of labelling regulations, it is likely to be subjected to more exhaustive tests than afforded by routine screening methods.

Implications for validity of GM food analysis by PCR

  33. Many foodstuffs which may need to be labelled will be subject to varying degrees of processing, frequently with the addition of ingredients which result in a complex food matrix. Detection of DNA by PCR will therefore probably be influenced by the relative extent of PCR inhibition and DNA degradation on a food by food basis.

  34. There are very significant challenges in the detection and quantification of processed foods and food components even with complete knowledge of the processing history of the sample, its origins and purity. The possibility of identification of processed food as containing GM source ingredients should ideally be elucidated on a case by case basis. Given the variety and complexity of foodstuffs available in today's markets this would pose a considerable expense for the industry.

  35. If absolute traceability of ingredients can be ensured during food preparation, then testing for genetic modification in less processed or raw ingredients which are being added into the food product would be a more reliable option for determining whether the final food product contains GM material. This can be achieved from the ingredients if traceability exists and, in fact, a wide range of related products could all be assessed from the smaller range of initial ingredients, saving time and money.


  36. In common with other food labelling regulations a de minimis threshold has been proposed for the labelling of GM foodstuffs, to potentially allow for the adventitious "contamination" of a foodstuff or ingredient through the food supply chain. Such a threshold, which has been considered at 1-3 per cent, in line with other food legislation, would necessitate quantitative analysis of [GM] foodstuffs.

  37. In consideration of a de minimis threshold for the presence of DNA [or protein] resulting from genetic modification the following issues should be carefully considered:

PCR detection limits

  38. Detection limits were determined for GM soya using serial dilutions of purified GM soya DNA with non GM soya DNA to stimulate different mixtures of GM and non GM soybeans. Detection limits were determined to increase from 1 per cent to 0.01 per cent as the extent of PCR amplification was increased above standard practise (Wurz and Willmund, 1997). At LGC we have also conducted experiments to determine the limit of detection for GM soya flour in admixture with non-GM flour. Using standard PCR methodology a 0.1 per cent level of GM soya flour DNA was detected.

  39. Food matrix effects on the relative limits of detection possible with PCR were demonstrated in experiments carried out by Greiner and Konietzny (1997). These researchers reported experiments in which they introduced "foreign" (E. coli) DNA into a baking process. DNA was extracted at different stages of processing, and PCR employed to detect the introduced E. coli DNA.
Experimental results demonstrating that the limit of detection by PCR
of "foreign DNA" is significantly increased by food processing

Processing stageLimit of detection by
PCR (gene "copies")

Pure DNA1
DNA added to rye flour100-150
Bakednot detected

  40. These results clearly demonstrate that at each successive stage of the baking process it becomes more difficult to detect the inserted "foreign DNA", even though the same percentage of DNA is present as an ingredient at the end of the process as at the start. It is expected that the same situation would apply in the case of detection of "foreign" GM soya flour in bakery products.

  41. Therefore, the evidence suggests that the ability to detect GM residues in food significantly decreases as the level of food processing increases. If the requirement in the proposed labelling regulations would be for a 1 per cent cut off for the particular food product, then in more highly processed foods which may actually contain a high percentage of for example GM soya, the level of detection may be below this threshold value, and the product could potentially be labelled "does not contain GM soya".

  42. There is therefore a requirement to establish the relative limits of detection (under standardised PCR conditions) for different types of foods, to inform the proposed legislation.

Quantification of amount of GM material by PCR

  43. Future EU legislation is likely to set limits for the percentage of GM material below which food can be labelled as not containing GM Derived material. Enforcement of such legislation will require development of testing procedures which can accurately measure the amount of GM material in food samples.

  44. Precise and accurate quantification of the amount of GM material in any given sample is analytically very demanding. However, this would be required for enforcement of labelling regulations, which would necessitate, the confident determination of the level of GM material in a food for instance, 0.5 per cent may be below the threshold value for labelling whereas 1.5 per cent may be above. Exact PCR quantitation is essentially still at the developmental stage for food analysis, and is not yet routinely possible.

  45. Current quantitative PCR methods are of two major types:

    (1)  Threshold detection analysis is really an extension of limits of detection analysis and involves PCR application of standards containing known amounts of GM material alongside unknown samples. Instead of looking for presence or absence of bands against known standards, the relative yields of amplified product from the standards and unknown samples are compared to enable quantitation.

    (2)  Quantitative competitive PCR (QCPCR) relies on the co-amplification of known amounts of a second DNA target or mimic. In this competitive amplification, both targets have the same PCR priming sites and compete for the available reagents in the PCR reaction. The result of the competitive nature of the reaction is that the relative yield of target and mimic can be related to the starting ratios of the two. By competing an unknown amount of one target with a known dilution series of a suitable mimic, the amount of target GM DNA can be calculated.

  46. An advantage of QCPCR is that amplification of mimic and target are carried out in the same tube providing an internal PCR control. The disadvantage of QCPCR is that development of PCR mimics can be technically challenging and new mimics will have to be generated for each new target.In contrast, quantitation using threshold detection analysis only requires access to the PCR primers used for standard detection and standard DNA samples of known composition.


LGC final report on MAFF project number 2B065 "The Detection of Genetically Modified Organisms in Food." (1998)

LGC partner reports to EU-project SMT4-CT96-2072 "Development of Methods to Identify Foods Produced by Means of Genetic Engineering." (1997, 1998)

Greiner, R and Konietzny, U (1997) Is there a possibility to identify processed foods as produced through genetic engineering by PCR technology? pp. 100-102 in Foods Produced by Means of Genetic Engineering. Second Status Report. Bg VV Hefte, Berlin.

Wurz, A and Willmund, R (1997) Identification of transgenic glyphosate-resistant soybeans pp. 115-117 in Foods Produced by Means of Genetic Engineering. Second Status Report. BgVV Hefte, Berlin.

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