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3.1 PCR-based methods

Although the principles of PCR had been conceptually described already in 1971 (Kleppe et al., 1971), experimental data were first published in the mid 1980s (Saiki et al., 1985). Since then, this technique has revolutionised molecular biology and many other areas in the bio-medical sciences. The number of references to PCR in the scientific literature has been estimated to be more than 40,000 (White, 1996). The high chemical and thermal stability of DNA, the high sensitivity of the method, its technical simplicity, the vast amount of experience already accumulated with it, along with the apparent potential for automation (Abramowitz, 1996; White, 1996) are main advantages of this method, establishing the current prevalence of PCR-based detection methods. This preference is likely to continue in the foreseeable future.

3.1.1 Officially validated identification methods

This section discusses methods which have been specifically developed to detect GMOs or products derived from GMOs in food stuffs and that have been included in a collection of official methods. Methods currently in the process of being validated will also be discussed.

The only official, validated methods that have been published so far, were developed by the BgVV-working group ('Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin', Berlin) for the 'development of methods to identify foods produced by means of genetic engineering' ('Entwicklung von Methoden zum Nachweis mit Hilfe gentechnischer Verfahren hergestellter Lebensmittel'). These methods have been included in the listing of official methods ('Amtliche Sammlung von Untersuchungsverfahren') according to Article 35 of the German Food Act (LMBG, Lebensmitel- und Bedarfsgegenständegesetz). Included among these methods were the results of inter-laboratory studies with participants from academic research institutes, private laboratories and food control authorities.

Three methods have been developed by this group, two of which have already been included in the list of official methods according to §35 LMBG. The two detection methods describe the PCR-based identification of a genetically engineered potato and a genetically modified microorganism in fermented raw sausages. A third assay, describing the detection of a genetically modified microorganism used as a starter culture in yoghurt, was tested in an inter-laboratory study in the end of 1996. All these methods have a model character since none of the utilized GMOs has been approved yet in any country and the use of any of these organisms in their current form in food is not intended. The methods for the detection of genetically engineered potatoes and genetically modified microorgansims in fermented raw sausages have recently been reviewed in the Bundesgesetzblatt (Schulze et al., 1996).

3.1.1.1 Genetically modified potatoes

PCR amplification of the altered DNA sequence and validation via DNA probe hybridisation has been used to identify a genetically engineered potato (LMBG-Methodensammlung, 1996; Schulze et al., 1996). The potatoes tested carried an introduced invertase gene from Saccharomyces cerevisiae and a transgene coding for a hygromycin phosphotransferase. The method is well documented; DNA extraction and DNA amplification by PCR were followed by separating the amplification product using agarose gel electrophoresis and controlling the length of the amplified product by size controls. After electrophoresis, the DNA was transferred onto membranes and analysed for the presence of the respective DNA sequence using DNA-DNA hybridisation (Southern-Blot). The specificity of the method was confirmed in inter-laboratory studies which yielded a reliability of more than 97 %. The amplicon (amplified DNA fragment) was 837 basepairs in length and contained sequences of the hygromycin phosphotransferase gene (Primer sequences and amplicon length in PCR-assays to detect GMOs). The specificity of the method is somewhat limited since any organism carrying this gene could be detected by the method.

3.1.1.2 Genetically engineered Lactobacillus in raw sausages

An analogous method developed by the BgVV working group was designed to identify genetically engineered Lactobacillus curvatus in raw sausages by PCR-based DNA amplification and hybridisation (LMBG-Methodensammlung, in press; Schulze et al., 1996). The Lactobacillus curvatus strain carried a plasmid-encoded catalase gene (katA) derived from Lactobacillus sake (Hertel et al., 1995a; Hammes and Hertel, 1996). The plasmid also carried a gene coding for chloramphenicol acetyl transferase (cat). Apart from its somewhat modified DNA extraction procedure, the method is similar to what was described for the potato. The reliability of the technique was determined in inter-laboratory studies to be more than 95 %. The primers used were complementary to sequences in the plasmid (in the cat gene) and to sequences in the katA gene, respectively, resulting in an amplicon of 1321 basepairs (Primer sequences and amplicon length in PCR-assays to detect GMOs). Since the amplicon contains the interface between plasmid sequences (cat) and the transgene (katA) this method should be highly specific for the genetically engineered microorganism that was used. Because the sequence of interest was located on a plasmid, the copy number of the sequence (and thus the sensitivity of the method) may be higher as compared to sequences that are integrated into the bacterial genome. The applicability of the method for strongly heat- or acid-treated samples may be limited due to the comparably large size of the amplicon chosen. (This subject will be dealt with in subsequent sections.)

3.1.1.3 Genetically engineered Streptococcus used a starter culture in yoghurt

Another model system for detecting a genetically modified microorganism was elaborated for Streptococcus thermophilus, a bacterial strain used as a starter culture in yoghurt (LMBG-Methodensammlung, in preparation). The method is analogous to the methods described before; again, the DNA extraction procedure was somewhat optimised (Lick et al., 1996a). Since certain results of the inter-laboratory studies were still unavailable as of January 1997; a precise assessment on its reliability can not yet be provided. The primers recognise sequences of the homologous lacZ gene and the (heterologous) chloramphenicol acetyl transferase (cat), which represents the transgene in this model-GMO (Heller, 1995). The amplicon used was 623 basepairs in size (Primer sequences and amplicon length in PCR-assays to detect GMOs) and contains an interface between homologous and heterologous sequences, ensuring high specificity of the method. As a positive control, species-specific PCR amplification of the lacZ sequences from Streptococcus thermophilus was used.