Interview with Dr Hermann Rüggeberg and Dr Lothar Kruse, Impetus Bioscience:
Genetically modified cotton seeds have been marketed since 1995. They contain genes that give them properties such as resistance to pests, e.g. the cotton bollworm, or herbicide tolerance. In Europe, the cultivation of genetically modified plants is almost only allowed in field trials. Food or animal feed which is to be labelled as “organic” or “eco” must comply with the requirements of EU Organic Regulation 834/2007, which precludes the use of genetically modified organisms. In the context of the increasing demand for transparency within the supply chains and to control advertising claims for products being offered, tests on genetically modified organisms, or GMOs, are also gaining importance in cotton textiles. We talked about this with Dr Hermann Rüggeberg and Dr Lothar Kruse, managing directors of the respected DNA analysis company Impetus Bioscience in Bremerhaven.
Cotton Report: Which molecular biological methods are used for the GMO or DNA analysis of cotton?
Hermann Rüggeberg: Impetus has 20 years of experience in detecting, identifying and quantifying genetic engineering changes in plants using a variety of methods. We have expanded our analytics to such an extent that not only known but also many unknown changes can be identified or detected. Of particular importance is the distinction between the least traces of genetically modified cotton and higher percentages. We carry out the necessary quantitative measurements exactly, which is only possible by using state-of-the-art real-time PCR technologies over many years of practice. Consecutive methods include screening, construct verification, and event identification.
What differentiates screening from construct verification and event identification?
Lothar Kruse: The genetic material (DNA) of all living organisms contains information-carrying sections (genes) and additional regulatory DNA sections, the so-called promoters and terminators, which define the beginning and the end of a gene. All EU-approved GM plants and most of the GM plants grown worldwide contain regulators, often using the same ones in a wide variety of plants. Thus, a positive screening result only indicates a genetic modification, which only allows a positive or negative diagnosis of GMOs.
A gene construct consists of one or more genes and the associated regulatory DNA sequences. Identical constructs have often been transformed into various plant species. The proof of a construct proves only a certain genetic change.
Event-specific detection identifies the exact transition region between the introduced construct and the host DNA. In contrast to screening and construct verification, which can only give indications of a genetic change, event identifications are considered as direct and allow the immediate unequivocal classification of the respective genetically modified plant. For example, specific DNA sections of genetically modified insect resistant cotton, e.g. with the event name MON 531 or MON 15985 can be identified and, if necessary, quantified.
How does the real-time PCR method work?
Lothar Kruse: The polymerase chain reaction is a method of making millions of identical copies of a defined section of DNA. Here, cycles are normally repeated 45 times, with DNA doubling in each cycle, i.e. there is an exponential reaction. In real-time PCR, this duplication can be seen online, that is in real-time. In addition, the quantitative determination of a duplicated DNA is possible. This method should not be mistaken for the reverse transcriptase polymerase chain reaction. The reverse transcription is a fundamentally different technique used in research and diagnostics, not to detect DNA, but RNA – another class of nucleic acids – found in cells, tissues or blood serum.
How does the procedure work?
Lothar Kruse: In the quantification of genetically modified DNA, two reactions are carried out in parallel. The first PCR duplicates a portion of DNA that occurs naturally in all cotton species. The second PCR amplifies a section of DNA specific for a particular genetically engineered cotton. If the signal strengths of the two reactions are identical, the proportion of genetically modified DNA is 100%. If the signal strengths differ, the genetically modified proportion is correspondingly lower. The measurement of the signals is based on fluorescent dyes which are incorporated into the newly formed DNA in each cycle of the PCR. The fluorescence increases proportionally with the amount of PCR products formed.
What are the requirements for a successful, objective test?
Hermann Rüggeberg: The basic requirements are that the laboratories are accredited, regularly participate in comparative laboratory tests and only use validated methods. For each sample to be examined, control reactions must be carried out to guarantee the certainty of results. Of course, it should also be shown with each sample whether it was possible to extract any DNA at all, because if no cotton DNA can be isolated, it is of course pointless searching for genetically modified DNA. Moreover, it is very helpful to have the appropriate range of methods available to be able to identify and quantify as many of the relevant genetically modified cotton lines as possible.
When is it difficult or impossible to find cotton DNA material?
Lothar Kruse: From cultivation to the finished product, cotton undergoes goes diverse processing methods in which the DNA becomes more and more fragmented. Mechanical processes pose no problems, unlike chemical stresses in certain dyeing or bleaching and finishing processes, for example with hydrogen peroxide. Here DNA is destroyed, i.e. its exact structure is no longer identifiable. In our experience, this is the case in about 10 percent of the samples. We therefore recommend sampling at an early point in time when the cotton is bought or arrives at the spinning mill. However, the sample should not only be taken from one point on the surface of a cotton bale, but from different points and possibly also from inside the bale. When inspecting samples in which the cotton has been further processed, the source DNA must be identifiable.
To what percentage can genetically modified components in cotton be found?
Hermann Rüggeberg: Based on our practical experience with the examination of samples of finished textiles by PCR, we consider a 0,1 percent limit to be unrealistic. There is no general limit of quantification and this is always dependent on the amount of DNA that can be isolated from a sample. As the degree of processing of a sample increases, so the limit of quantification always decreases, i.e. the sensitivity decreases. The optimisation of DNA extraction procedures may lead to higher yields of DNA, but this will not change the fact that unprocessed materials are more sensitive to examination than final products. Nevertheless, the latest developments in our laboratory now also allow the examination of such end products which up to now could not be analysed.
What impact does contamination or mixtures of non-GM cotton and genetically modified cotton during processing have on test results?
Lothar Kruse: Mixtures or contamination of cotton of any kind occur in cultivation, harvesting and intermediate storage, in ginning, storage or even in spinning. Here, cotton of different origins is often mixed, e.g. for the production of certain yarn qualities with special properties. At all stages, where a product must be produced and provided to one hundred percent from organic cotton, it is necessary to work as accurately as possible. The respective materials must be processed separately in cleaned machines. Precisely because it is so difficult to avoid carry-over, it is very important to be able to make reliable and meaningful quantitative statements. Because for customers, it is of course interesting to know whether genetically modified cotton is present more at the trace level, or in the high percentage range. In the first case, these are probably technically unavoidable carry-overs, in the latter case intent may play a role. To prevent irritation, it would be useful to think about a limit, below which small carry-overs are accepted. A pragmatic solution could be based on the Textile Labelling Act, which tolerates up to 2% of other animal fibres than the species declared in the product.
In your opinion, do you think that all testing laboratories test GMOs using the same method? What quality do the results have?
Hermann Rüggeberg: In principle, there are uniform methods, which however lead to fluctuating results in practice, depending on the way the laboratories operate. We therefore recommend that all relevant laboratories submit to a benchmarking test organised by a neutral body, where the results of tests on the same materials and their fluctuation ranges can be made comparable and harmonised. This is especially important when it comes to checking the validity of advertising claims.
In this context, it should also be mentioned that Impetus Bioscience has been working in close cooperation with ICA Bremen on GMO issues and in DNA analyses and from the testing practice we are striving for the definition of tolerable threshold values for GMO residues.
These threshold values could then be used as a basis for evaluation in international arbitration proceedings, for which Impetus Bioscience and ICA Bremen would already be able to carry out instrumental tests. A proposal for a universally acceptable tolerance level would eliminate the current contractual uncertainties and promote the proliferation of organic products similar to within the food sector.
Thank you for the informative and enlightening conversation!
Interviews embody the opinion of the respective interview partner and do not represent the position of the Bremen Cotton Exchange as neutral, independent institution.