Food authenticity is presently a subject of great concern to food authorities, as the incorrect labelling of foodstuffs can represent a commercial fraud. The implication of misleading labelling can be much more important concerning the presence of potentially allergenic foods. The need to support food labelling has provided the development of analytical techniques for the analysis of food ingredients. In the last years, several methods based on polymerase chain reaction (PCR) have been proposed as useful means for identifying species of origin in foods, as well as food allergens and genetically modified organisms (GMO), due to their high specificity and sensitivity, as well as rapid processing time and low cost. This work intends to provide an updated and extensive overview on the PCR-based methods for food authentication, including also methods for allergens and GMO the detection in foods.

Food authentication by PCR-based methods

Testing for genetically modified organisms (GMOs): Past, present and future perspectives.

Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed. GMO Detection method Database (GMDD) has collected almost all the previous developed and reported GMOs detection methods, which have been grouped by different strategies (screen-, gene-, construct-, and event-specific), and also provide a user-friendly search service of the detection methods by GMO event name, exogenous gene, or protein information, etc. In this database, users can obtain the sequences of exogenous integration, which will facilitate PCR primers and probes design. Also the information on endogenous genes, certified reference materials, reference molecules, and the validation status of developed methods is included in this database. Furthermore, registered users can also submit new detection methods and sequences to this database, and the newly submitted information will be released soon after being checked. GMDD contains comprehensive information of GMO detection methods. The database will make the GMOs analysis much easier.

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GMDD: a database of GMO detection methods

Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.

http://chglib.icp.ac.ru/subjex/2011/pdf/Anal.bioanal.Chem/Anal.bioanal.Chem.%202008,%20v.392,p.347-354.PDF

Advances in molecular techniques for the detection and quantification of genetically modified organisms

With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.

Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule.

Qualitative and quantitative analytical methods were developed for the new event of genetically modified (GM) maize, MON863 One specific primer pair was designed for the qualitative polymerase chain reaction (PCR) method The specificity and sensitivity of the designed primers were confirmed PCR was performed on genomic DNAs extracted from MON863, other GM events, and cereal crops Single PCR product was obtained from MON863 by the designed primer pair Eight test samples including GM maize MON863 were prepared at 001∼10% levels and analyzed by PCR Limit of detection of the method was 001% for GM maize MON863 On the other hand, another specific primer pair and probe were also designed for quantitative method using a real-time polymerase chain reaction As a reference molecule, a plasmid was constructed from a taxon-specific DNA sequence for maize, a universal sequence for a cauliflower mosaic virus (CaMV) 35S promoter used in most genetically modified organisms, and a construct-specific DNA sequence

Dong-Myung Min

Papers

Qualitative and quantitative polymerase chain reaction analysis for genetically modified maize MON863.