Foods Derived from Genetically Modified Organisms and Detection Methods by Wolfram Hemmer BATSThis report was prepared in close collaboration with the Swiss Federal Office of Public Health Table of Contents Summary................................................................................................................. 2 1 Introduction .......................................................................................................... 3 1.1 Field trials.......................................................................................................... 4 1.2 Transformation methods and genetic elements introduced into transgenic plants ...................................................................................................................... 7 1.3 Detection methods .......................................................................................... 10 2 GMO Products ................................................................................................... 12 2.1 Regulations ..................................................................................................... 12 2.1.1 Switzerland .................................................................................................. 13 2.1.2 The European Community ........................................................................... 13 2.1.3 USA.............................................................................................................. 14 2.2 Commercialisation of genetically modified products........................................ 15 2.3 Approval of genetically engineered products .................................................. 15 2.3.1 Differences in national approvals of the 'same' products ............................. 16 2.3.2 Summaries of national and multi-national approvals of genetically engineered products ................................................................................................................ 19 2.3.3 Restrictions .................................................................................................. 20 2.3.4 Commercially available products ................................................................. 20 2.3.5 Transgenic organisms in China.................................................................... 21 2.4 Enzymes derived by recombinant technology................................................. 21 2.5 Genetically modified animals .......................................................................... 22 3 Methods for identifying genetically engineered foods ........................................ 23 3.1 PCR-based methods....................................................................................... 23 3.1.1 Officially validated identification methods..................................................... 24 3.1.1.1 Genetically modified potatoes ................................................................... 24 3.1.1.2 Genetically engineered Lactobacillus in raw sausages ............................. 25 3.1.1.3 Genetically engineered Streptococcus used a starter culture in yoghurt... 25 3.1.2 Methods developed to detect GMOs and published in scientific journals..... 25 3.1.3 Highly specialised reports on the detection of GMOs in food unavailable in databases ............................................................................................................. 26 3.1.4 PCR diagnostics - problems and possible solutions in application............... 27 3.1.4.1 Choice of primers and general methodological parameters ...................... 27 3.1.4.2 Avoiding false-positive and false-negative results..................................... 28 3.1.4.3 Sensitivity.................................................................................................. 29 3.1.4.4 DNA quality ............................................................................................... 29 3.1.4.5 PCR methods applied to processed foods ................................................ 29 3.1.4.6 Inhibition of PCR and DNA extraction procedures .................................... 30 3.1.4.7 Verification of PCR results ........................................................................ 30 3.1.4.8 Reviews on the application of PCR in other areas .................................... 31 3.2 Various nucleotide-based amplification methods and their applicability.......... 31 3.2.1 Ligase Chain Reaction (LCR) ...................................................................... 31 3.2.2 Nucleic Acid Sequence-Based Amplification (NASBA) ................................ 31 3.2.3 'Self-sustained sequence replication' (3SR) and 'Q replicase amplification'. 32 3.2.4 Fingerprinting techniques (RFLP, AFLP, RAPD, etc.).................................. 32 3.2.5 Probe hybridisation ...................................................................................... 32 3.3 Protein-based methods ................................................................................... 33 3.4 Detection of enzymatic activities ..................................................................... 33 4 Genetic elements used in approved genetically modified agricultural crops - implications for screening and product-specific detection methods ...................... 34 4.1. Survey of genetic elements introduced into approved transgenic crops ........ 34 4.1.1 Survey of the structural genes used............................................................. 34 4.1.2 Survey of the promoters used ...................................................................... 36 4.1.3 Survey of the terminators used .................................................................... 37 4.2 Development of screening methods................................................................ 38 Table 19: How many PCR systems are needed? ................................................. 39 4.3 Product-specific detection methods and available sequence information ....... 40 5 National research projects devoted to the detection of GMOs........................... 40 5.1 The European Community .............................................................................. 41 5.1.1 Germany ...................................................................................................... 41 5.1.2 The United Kingdom .................................................................................... 41 5.1.3 The Netherlands........................................................................................... 42 5.2 Switzerland ..................................................................................................... 42 5.3 Canada ........................................................................................................... 42 6 Laboratories for food control in Switzerland....................................................... 42 7 Prospects ........................................................................................................... 43 8 GLOSSARY ....................................................................................................... 43 9 References......................................................................................................... 48 Summary This report reviews currently approved genetically modified food crops, products from modified microorganisms for food use, and molecular methods currently applicable or under development for detecting foods derived from genetically modified organisms (GMOs). Up-to-date reviews on approvals of genetically engineered organisms or food products (in the United States, the United Kingdom, the Netherlands, Australia, Canada, Japan, the European Community, and Switzerland), including a comprehensive compilation of genetic and biochemical information on the respective products are presented. >From a survey of the genetically engineered agricultural crops it was found that there were 28 approved, genetically distinct plant products (Minor genetic differences between several lines of certain products derived from independent transformation events of the same or virtually the same host plants were not considered here.). These products were chicory (1), corn (6), cotton (4), papaya (1), potato (2), rapeseed (4), soybean (2), squash (2), tobacco (1) and tomato (5). The survey on the genetic elements (promoters, structural genes, terminators) introduced into the approved modified agricultural crops along with other pertinent data presented in this report, could provide the basis for the development of efficient screening methods and product-specific techniques for detecting genetically engineered food products. The other main objective of this paper is to review published analytical methods developed for identifying genetically engineered foods. Relevant methods which may be useful for designing identification techniques are also given. Included are: validated, official methods for the detection of GMOs; methods developed for the identification of GMOs in food stuffs published in specialised scientific journals or in reports unavailable in databases; other scientific articles describing the identification of DNA sequences that have also been used for generating GMOs; and publications from relevant, related fields such as authenticity testing or the detection of pathogens. Most of the techniques currently available use the polymerase chain reaction (PCR) for the amplification of DNA-sequences introduced into the plants by genetic engineering. The applicability of other nucleotide-based