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Novel methods for the detection of GM materials in foods and food ingredients
Project Code: G01025
Current methods for the detection and quantification of Genetically Modified (GM) ingredients are predominantly ELISA and PCR based techniques that are now relatively standard analytical practice. However, the increasing demands placed upon trace nucleic acid detection technologies in other fields, such as medical diagnostics and forensic analysis, have driven the development and application of alternative, novel technologies that have the potential to reliably detect single target nucleic acid molecules within challenging matrices. This need for ultra-sensitive detection is in part driven by current trends in regulation, where limits are often set close to or even below the levels reliably detected by available measurement technology. The European Union (EU) 0.9 % limit for the detection of GM ingredients in potentially processed foods is a good example of where measurement struggles to match regulatory demands. However, such techniques have seldom been applied to issues of food authenticity and trace detection. This project therefore sought to examine and develop the application of two of the most promising emerging technologies in the context of the identification and quantification of GM ingredients.
One arm of the project was to assess the feasibility of using Rolling Circle Amplification (RCA) as an alternative amplification technique to PCR, for robust trace detection of GM material. The primary advantage of this technique is the scope for quantification, given that the RCA product is generated linearly over time. Quantification may be achieved by constructing standard curves of the RCA yield from assays with known input DNA concentrations. Another advantage of RCA is the ability to perform amplification of the target DNA under isothermal conditions, thus making the need for a thermal cycler redundant. The technique is also reputed to convey a 10-fold increase in sensitivity compared with conventional amplification via PCR. The second arm of the project (Part Two) sought to use mass spectrometry for the detection of GM ingredients (please see report number LGC/FA/2005/004)
For RCA based detection, using model systems involving both synthetic oligonucleotides and PCR products we were able to distinguish between GM and wild type motifs. However, when the assay was switched to real genomic targets this discrimination could not be achieved despite many attempts to modify reaction conditions. This is in line with the majority of published examples that to date have only used model systems and have not demonstrated feasibility using real genomic templates. We would therefore exercise caution in implying that model systems using PCR amplicons or synthetic oligonucleotides can illustrate the proof of concept for amplification by RCA of a padlock probe involving the use of genomic DNA samples.
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