Development and application of DNA analytical methods for the detection of GMOs in food

The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products.

Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil.

If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker.

Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity.

Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers.     

A general multiplex-PCR assay for the general detection of genetically modified soya and maize

The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for the detection of GMOs are necessary in order to verify compliance with labelling requirements. In the past few years, different PCR-based methods for the specific detection of the most economically important GMOs have been proposed. A molecular screening method based on multiplex-PCR that involves amplification of specific soya or maize sequences from plant DNA and the amplification of 35S promoter and NOS terminator for the detection of genetically modified soya and maize was developed. The m-PCR assay discriminated the GMO very quickly, reproducibly and in a cost saving and less time-consuming way. It is a flexible assay to conduce a preliminary GMO screening for detection of genetically modified soya and maize.     

Quantitative analysis of genetically modified organisms (GMO) in processed food by PCR-based methods

Two different PCR-based approaches for the quantitative analysis of genetically modified organism (GMO) – components in foods are presented using Soybean derived samples as an example. The first method – a double competitive PCR – is well suited to determine threshold levels of GMO content in food. The other – PCR on-line measurement – is suited to determine ratios of transgenic versus non-transgenic component. Both methods provide a means to alleviate the problems of standardisation encountered with simple qualitative PCR approaches and will allow to cope with threshold levels for GMO, once issued by legislative bodies.     

Results of an interlaboratory assessment of a screening method of genetically modified organisms in soy beans and maize

The preliminary results on an interlaboratory trial on the detection of genetically modified organisms (GMO) are presented. The method applied is based on the detection of modified DNA using the polymerase chain reaction (PCR) for amplification. The amplified fragments analysed are derived from the 35S promotor and the NOS terminator used for modification and are present in 26 from the 28 GMOs currently already approved or under approval by the competent authorities. This method fits as a screening method indicating the presence of GMO in food. However, it does not allow an identification of the kind of GMO present in the samples. Samples of soybean and maize flour containing 0%, 0.1%, 0.5% and 2% GMO had been prepared for this study and are also already commercially available. In this paper the combined results from 27 laboratories are presented, indicating that on average the probability of false positive or false negative results is only about 1% for soybeans and below 5% for maize.     

Fluorophore double stranded probe-multiplex quantitative PCR method for detecting transgenic component of promoter derived from Cauliflower Mosaic Virus and nos terminator derived from Agrobacterium tumefaciens simultaneously

The article is to establish multiplex PCR method for quantitative detecting transgenic component promoter derived from Cauliflower Mosaic Virus (CaMV 35S) and nos terminator derived from Agrobacterium tumefaciens (Tnos) in foods. According to the specific sequence of CaMV 35S and Tnos which have been used in genetically modified organisms (GMOs) frequently, and the sequence of soybean endogenous lectin gene, three pairs of primers and corresponding fluorophore double stranded probes (FDSP) were designed to allow for quantitative detecting of GMOs. FDSP designed with maximal specificity also showed the greatest detection sensitivity, and the ease in design, the simple single-dye labeling chemistry. FDSP-multiplex quantitative PCR (FDSP-MQPCR) methods were established for the detection of transgenic component CaMV 35S and Tnos simultaneously. Ten soybean flour samples were tested with FDSP-MQPCR method. The method gives five positive-samples with quantitative results in 5 h, and accuracy rate is above 97.0%. The described methods enabled a sensitive, specific, simple, and accurate detection of transgenic component and thus provide a useful tool for quantitative analysis of raw and processed food products. FDSP-MQPCR method has not only improved detection efficiency and result credibility, but also has guaranteed the better accuracy and repetitiveness.     

Disposable genosensor, a new tool for the detection of NOS-terminator, a genetic element present in GMOs

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. An indicator based electrochemical disposable genosensor for the voltammetric detection of NOS-terminator, a genetic element present in GMOs is described as a possible substitute method for the common technique of gel electrophoresis and fluorescent image analysis. The biosensor relies on the immobilization of the 25-mer single stranded oligonucleotides (probe) related to NOS-terminator DNA sequence and the relative binding of this sequence with the polymerase chain reaction (PCR) amplified samples from certified reference material (CRM) of Roundup Ready soybean (Fluka) at a screen printed electrode (SPE). The extent of hybridization between the probe and target DNA is determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB), as the hybridization indicator. The difference between the MB signals, obtained from the hybrid modified and probe modified SPEs, is used to detect GMOs from PCR amplified DNA samples. Numerous factors affecting the hybridization and indicator binding reactions are optimized to maximize the sensitivity.     

Applicability of quantitative PCR to soy processed foods containing Roundup Ready Soy

Detection methods for genetically modified organisms (GMO) have been developed among countries, but there are still no practical quantitative methods for the processed foods. Although the quantitative methods in the Japanese standard are now targeted at raw grain, we examined whether the methods may also be applied to processed soy foods. The Roundup Ready Soy (RRS) in soy products by our own or professional making were quantified using real time PCR, and we compared the RRS content with that in raw soybeans. As a result, sufficient copy numbers of the lectin and introduced gene were obtained for quantification from all the soy products. Almost all items showed no significant differences in the RRS content. These findings suggested that the Japanese official quantitative method might be useful for screening in some soy products.     

Membrane based detection of genetically modified organisms in some representatives food

Recently, DNA-based techniques became very common for the detection of genetically modified organisms (GMOs) in food products. For rapid and easy detection of GMOs, polymerase chain reaction (PCR) screening methods, which amplify common transgenic elements, are applied in routine analysis. Incorporation of PCR and membrane method introduced in this study offer an alternative detection of GMOs. In this study, a total of 32 samples and three certified reference materials were tested for the existence of the 35S promoter of cauliflower mosaic virus (CaMV) and 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) gene residues. Dot blot screening system introduced in this study can be routinely used as a semi-quantitative screening of GMOs.     

Detection methods for genetically modified crops

Due to the market introduction of genetically modified crops (GMOs) as the Roundup Ready (RR) soya and Bt corn, the European food industry came face to face with the question of the use and labeling requirements on GMO crops and its derivatives. Although even today, no defined European legislation is available, a definitive need for detection methods exists. Both DNA and protein based methods have been developed and applied for the detection of RR soya beans and its derivatives. For the CP4 synthase, synthetic peptides corresponding with the antigenic and non-homologous parts of the CP4 synthase were synthesized and mono-specific anti-CP4 synthase monoclonal antibodies were prepared by hybridoma technology. The monoclonal antibodies were able to detect the CP4 synthase in the RR soya using Western blotting analysis. Detection limits were found between 0.5% and 1%. The method is currently validated for half-and final products. The applied DNA methodology was making use of polymerase chain reactions (PCR) using sets of primers along the gene encoding the Agrobacterium CP4 synthase. DNA extraction and purification conditions were examined on a case-by-case approach for a scala of soya products (lecithin, oil, soybean meal, soy protein isolates etc.), half-products and final consumer products. Detection limits were found between 0.01% and 0.1%. In this paper a comparison will be made between the two types of methods in relation to sample preparation, sensitivity, validation and the use for half-products and final consumer products.     

Transboundary movement of genetically modified organisms in India: Current scenario and a decision support system

Genetically modified (GM) crops have benefited global agriculture by introduction of superior traits for better agronomic performance, ensuring nutritional security and mitigating climate change. In India, to meet the demand of burgeoning population and to withstand the changing climate, GM crops would play an important role. Since 1997, GM crops are being imported through Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, the designated nodal organization for quarantine processing and import of GMOs (referred to GM planting material in present context) for research purposes. In the present study, an attempt has been made to analyze the trend of import of GMOs. Till the end of 2015, 205 consignments of fifteen GM crops have been imported from 19 countries by public and private sector. Detailed analysis of diversity in traits of imported GM events and imported stacked traits in cotton and maize has been made. In the recent past, four consignments of GMOs have been exported for research purposes. Involvement of public/private sector in transboundary movement of GMOs was evaluated. Along with quarantine processing of imported/exported GMOs, molecular testing for specific transgenic elements as claimed by the importer/exporter is also carried out employing polymerase chain reaction (PCR) and real-time PCR based markers. Efficient detection strategies based on GMO matrix as a decision support system, loop-mediated isothermal amplification and multi-target real-time PCR-based systems have been developed. The data presented herein would provide a decision support system to check for authorized/unauthorized GMOs in food and supply chain.     

Detection of GMO in food products in Brazil: the INCQS experience

Regulations for the use and labeling of genetically modified organism products and derived ingredients are being implemented worldwide, what demands reliable and accurate methods to detect genetically modified organisms (GMO) in raw materials and food products. This study aimed at monitoring products derived from GMO in the Brazilian market using detection methods for the presence of Roundup Ready soybean, Bt176 and MON 810 maize. The results demonstrate for the first time the presence of GM-soy in Brazilian food products, reinforcing the need for the development of accurate quantitative methods in routine analyses.     

A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean,maize and rice in highly processed products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of the GMOs are necessary in order to verify compliance with labeling requirements. There are few effective screening methods for highly processed GM (genetically modified) products. Four genes (CP4-EPSPS, Cry1A(b), BAR, and, PAT) are common exogenous genes used in commercialized transgenic soybean, maize, and rice. In the present study, a multiplex nested polymerase chain reaction (PCR) method was developed to simultaneously detect the four exogenous genes and one endogenous gene in two runs. We tested eleven representative highly processed products samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, soybean refine oil, soybean salad oil, maize oil, maize protein powder, maize starch, maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products except for soybean refined oil, soybean salad oil and maize oil, and the sensitivity was 0.005%. These results indicate that multiplex nested PCR is appropriate for qualitative detection of transgenic soybean, maize and rice in highly processed products except for refined oil.     

Challenges for methods to detect genetically modified DNA in foods

Qualitative detection methods for genetically modified (GM) DNA sequences in foods have evolved fast during the past years. The sensitivity of these systems is extremely high, even for processed foodstuffs. However, in future, quantitative results about the fraction of GM material in a composite food will be needed and the fast increasing number of GM foods on the market demands the development of more advanced multi-detection systems. Other challenges and problems might arise from the decreasing relevance of methods which screen for sequences commonly found in GMOs, the inability to detect GM foods for which the modified sequence is unknown, the lengthy standardisation procedures and the need to up-date continuously databases comprising commercially available GM foods and the respective detection strategies.     

Event-specific qualitative and quantitative PCR detection of LY038 maize in mixed samples

With the development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative PCR detection methods have become the internationally agreed state-of-art. This paper describes the characterization and event-specific quantitative detection of LY038 maize insert with the application of reference molecule. The flanking regions were characterized by Inverse-PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right flanking sequence. In the qualitative PCR assay, two PCR systems were established with the event-specific and specie-specific primers respectively, and the limit of detection (LOD) was 0.1% (approximates to 37 haploid genome copies). In the quantitative TaqMan real-time PCR assay, a reference molecule was constructed by recombinant PCR and standard curves were set up. By using the reference molecule, we obtained standard curves with good linearity and relatively high efficiency of PCR reaction. The results indicated the usability of the plasmid as standard material. From above results, we believed that the established event-specific qualitative and quantitative PCR systems for LY038 maize in this study were acceptable and suitable for LY038 maize detection in mixed samples.     

Comparison of simplex and duplex real-time PCR for the quantification of GMO in maize and soybean

This paper focuses on the determination of the GMO content of maize and soybean samples using real-time PCR, comparing simplex and duplex PCR. The total DNA content of samples was determined by amplifying part of a maize gene encoding a lipid transfer protein, or part of a soybean lectin gene. The transgenic DNA was quantified by amplifying part of the CaMV 35S promoter. The importance of preparation and conservation of standards as well as the relevance of DNA extraction protocol on the variability of results are discussed. For the determination of low GMO content, limitation in the number of copies of the target gene to be amplified is considered. For samples with a theoretical GMO content of 1%, corresponding to the legal threshold for labelling, the value determined by duplex real-time PCR ranged from 0.85% to 1.20%. Both real-time simplex and duplex PCRs allowed identification of GMO free samples without ambiguity.     

Evaluation of a rapid DNA extraction method to detect yeast cells by PCR in orange juice

Yeasts are the main causes of spoilage in pasteurized orange juice. Whereas detection by plate counting techniques is too slow to allow appropriate intervention measures, PCR reaction offers a rapid alternative, but it can be inhibited by components of food samples. We developed a DNA extraction method directly from orange juice for rapid yeast detection by PCR amplification of the rRNA internal transcribed spacers including the 5.8S rRNA gene at a detection limit of 10³ cfu/ml juice sample. We show that it is possible to reduce detection time and improve detection rate of yeasts in orange juice by using a simple glass bead disruption procedure in connection with silica absorption and amplification technology.     

SIGMO: A decision support System for Identification of genetically modified food or feed products

Since their introduction in 1994, more and more genetically modified (GM) crops are grown worldwide and introduced in food or feed products. In the European Union (EU), the production, trade and marketing of GM products is strictly regulated, but the situation is becoming more complex due to the increasing number and complexity of GM crops, and asynchronic approval procedures with the major GM crop producing countries. Importers and traders are obliged to assess their respective supply chains for the potential presence of authorised and unauthorised GM organisms (GMOs), where wrong decisions may lead to substantial economic losses. This article presents a decision support system SIGMO aimed at guiding producers and traders with the assessment of the likelihood that their products may comprise authorised or unauthorised GM materials. The assessment is based on traceability data about the product (nature and origin of the raw materials, transportation aspects), as well as analytical results of the presence of GMOs in the final product or its ingredients. The approach uses a combination of data-driven and model-driven decision support. SIGMO is composed of (1) a data base providing data about GMO crop species produced and approved in counties worldwide, (2) a multi-attribute model for the assessment of GMO presence in food/feed products, and (3) an on-line user interface. SIGMO helps producers and traders to better comply to valid EU GMO regulations and to better control their products and supply chains in terms of the unintended presence of (unauthorised) GMOs in a cost-effective way.     

Prevalence of genetically modified foods (GM foods) in the United Arab Emirates

The real and/or perceived risks of genetically modified organisms (GMO) prompted food safety regulators to label the GM products. Although there are no legislations on GM labeling and cultivation of GM crops in the UAE, the present study aims to monitor the status of GM foods in the UAE market using Light cycler real time PCR technology and GMO screening kit. The yield and purity of DNA extracted by CTAB method was higher when compared to Qiagen plant kit with an exception of soya products for which Qiagen kit yielded better results.Out of 128 samples tested, 16 were positive for plant, 35S promoter and Tnos fragment. In conclusion, GMO screening assay applied in this study confirms the presence of genetically modified food in the UAE market. The rapidly growing GM market with multiple events and the threat from unapproved events signifies the value of surveillance program for monitoring the status of GM foods.     

Applicability of a novel reference molecule suitable for event-specific detections of maize NK603 based on both 5′ and 3′ flanking sequences

Plasmid molecule based reference material (RM) has been shown to be a good alternative as the calibrator for genetically modified organisms (GMOs) identification and quantification, while most of the currently developed plasmid RM can only be used for one specific target detection. In this study, a flexible plasmid RM pNK containing three DNA fragments, i.e. 5′ and 3′ event-specific sequences of maize NK603 and endogenous gene zSSIIb, was developed. We have proved that pNK is suitable for using as a calibrator in both 5′ and 3′ event-specific detection of maize NK603, compared with that of genuine genomic DNA. The limit of detection (LOD) was 10 copies of pNK DNA in conventional PCR assays. The absolute LOD and limit of quantification (LOQ) in quantitative PCR assays were 5 and 25 copies. The standard curves targeting to zSSIIb, 5′ and 3′ event-specific sequences based on pNK DNA showed high reaction efficiency and good linearity. Also, low bias and variations were obtained in practical samples quantification using pNK as the calibrator. These results demonstrated that the developed pNK is flexible and suitable for identification and quantification of maize NK603, as a preferable substitute of RM from the plant raw material.     

Detection of Roundup Ready soybean by loop-mediated isothermal amplification combined with a lateral-flow dipstick

LAMP–LFD, loop-mediated isothermal amplification (LAMP) combined with a lateral-flow dipstick (LFD), was developed and evaluated as a new method for the detection of Roundup Ready soybean (RRS). Biotinylated LAMP amplicons were produced by two sets of six designed primers that specifically recognized the endogenous gene (Lec1) and the event-specific 5′ -junction region (G35S) of RRS followed by hybridization with FITC-labeled probes and LFD detection. The following optimized conditions for the LAMP assay were used: deoxynucleotide triphosphate (dNTP) concentrations ranging from 0.6 to 3.2 mM, 6 mM Mg²⁺, 4 U Bst DNA polymerase and a 1:6 ratio of outer to inner primers. The LAMP–LFD results were generated within 50 min. The detection limit of LAMP–LFD was 2.4 copies of the linearized plasmid pTLH10 and was 20 times more sensitive than conventional PCR. We demonstrated the high specificity of LAMP–LFD by testing processed soybean products, genetically modified (GM) maize and Bt-cotton meal. The novel LAMP–LFD setup presented here is simple, rapid, and has the potential for future use in the detection of GM ingredients in feed and food products.