Development of an event-specific assay for the qualitative and quantitative detection of the genetically modified flax CDC Triffid (FP967)

The genetically modified flax, event FP967, with tolerance to sulfonylurea herbicides, is one of the commercial genetically modified events approved in Canada and USA.This event is not authorized in Switzerland and EU, therefore, a method to specifically detect the CDC Triffid line, was required. We revealed the 3′ integration junction sequence between host plant DNA and the integrated gene construct FP967 by means of Restriction Site PCR and both a qualitative and a quantitative PCR detection assays were developed. The qualitative PCR revealed a limit of detection of 0.01% of GM flax in 100 ng of genomic DNA. The quantitative PCR assay showed a limit of detection of about 9 haploid genome copies. The specificity and sensitivity of the assay indicate that the developed event-specific PCR methods can be used for identification and quantification of FP967 flax.     

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.     

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.     

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 sixteen genetically modified maize events in processed foods using four event-specific pentaplex PCR systems

To efficiently identify genetically modified (GM) maize events in foods and feeds, we report here the development of four individual pentaplex PCR analysis systems for event-specific identification of sixteen GM maize events approved in South Korea. In addition to the maize endogenous reference gene, zSSIIb, the four pentaplex PCR assays target group 1 containing TC6275, MON810, T25, and NK603; group 2 with TC1507, MON863, GA21, and DAS-59122-7; group 3 with MIR604, Bt11, Bt176, and MON89034; group 4 with event 3272, LY038, MIR162, and MON88017. These amplicons were designed to be smaller than 200 bp to make the testing system suitable for analyzing processed foods/feeds derived from these 16 GM maize events. After optimizing the reaction condition, the limits of detection of these four assays were approximately 0.25% for all of the 16 GM maize events. This multiplex PCR method for sixteen GM maize events was validated by three operators, and the data confirmed the reliability of the developed assays. Furthermore, 74 food samples containing maize ingredients from the USA, China, Japan, and South Korea were analyzed, and we observed 18 food products containing one or more GM maize events. These results suggest that the developed multiplex system is applicable for use in specific testing of sixteen GM maize events in foods and feeds in South Korean market.     

Quantitative competitive PCR for the detection of genetically modified organisms in food

Present polymerase chain reaction (PCR) detection methods only allow the qualitative detection of GMO in food without quantitation of the GMO content. Clearly, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. This quantitative competitive (QC) PCR was first described in the early nineties and is widely used nowadays.

We have developed and evaluated QC–PCR systems for the quantitative detection of Roundup Ready^TM soybean (RRS) and Maximizer maize (MM) in food samples. Three DNA fragments differing from the GMO specific sequences by DNA insertions were constructed and used as internal standards in QC–PCR. These standards were calibrated by co-amplifying with mixtures containing defined amounts of RRS DNA and MM DNA, respectively. The calibrated QC–PCR systems were applied to several commercial food samples containing RRS and to three certified RRS flour mixtures (Fluka standards). Recently, quantitative methods for the detection of RRS were successfully tested in a collaborative study involving twelve European control laboratories. Thus, QC–PCR methods will allow to survey “de minimis thresholds” of GMOs in food.     

Development of triplex PCR for simultaneous detection of maize,wheat and soybean

A reliable and fast detection of important food plants, such as maize (Zea mays L.), wheat (Triticum aestivum L.), and soybean (Glycine max L.) is of particular interest for food authenticity and safety assessment. In this study, the novel multiplex polymerase chain reaction (PCR) method was developed for the rapid qualitative detection of soybean, maize and wheat. To this purpose, new soybean-specific and maize-specific PCR primers were designed. Their specificity was assayed by uniplex PCRs with different plant species, namely maize, soybean, wheat, oats (Avena sativa), and barley (Hordeum vulgare L). Gel electrophoresis of the amplification products demonstrated high specificity of both primer pairs for identification of relevant species. Subsequently, based on the developed DNA markers, the species-specific triplex PCR targeting maize invertase gene, soybean lectin gene and wheat low-molecular-weight glutenin subunit was developed and optimized for simultaneous identification of these three plant species. The developed PCR method enables specific, effective and rapid detection of maize, wheat and soybean and may be used for food analysis.     

Applicability assessment of a real-time PCR assay for the specific detection of bovine,ovine and caprine material in feedstuffs

A TaqMan real-time polymerase chain reaction (PCR) method was developed for specific detection of bovine, ovine and caprine processed animal protein (PAP) in industrial feedstuffs. The method uses species-specific primers and probes targeting short mitochondrial D-loop sequences, and a positive amplification control based on 18S rRNA gene. The applicability of the real-time PCR protocol was assessed through analysis of 126 industrial feed samples that were manufactured to reproduce rendering processes of commercial feeds destined for farmed animals. The assay successfully classified samples as positive or negative according to the ruminant composition, enabling qualitative detection of banned material in feeds at levels as low as 0.1%. Although the method provides quantitative potential, results suggest that the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing treatments of the feeds, which affect the amount and quality of amplifiable DNA.     

Development of a multiplex PCR method for testing six GM soybean events

Genetically modified (GM) soybean and derived products make up a large part of the biotech-derived food and feed market. As more GM soybean varieties have been approved for commercialization, labeling requirement by South Korea and other countries needs the technical testing methods. This paper reports the development of a multiplex PCR method for identifying six commercialized GM soybean events using the event-specific fragment. Event specific primers targeting Roundup Ready Soybean (RRS, GTS40-3-2), A2704-12, DP356043-5, MON89788, A5547-127, and DP305423-1 were designed, and a multiplex PCR assay consisting of six event-specific fragments and one endogenous lectin fragment was developed. The specificity of the event-specific PCR method was confirmed using 20 GM events of maize, soybean, cotton, and canola. The limit of detection (LOD) for each event in the multiplex PCR is approximately 0.05%. Intra-lab validation by two different operators confirmed the specificity and LOD of this multiplex PCR method. The method was used to test 30 soybean-derived foods from South Korean and US markets, and results revealed three varieties of GM soybean (RRS, A2704-12, and MON89788) in 19 of the 30 food samples tested. This work provides an efficient and cost-effective approach for event-specific analysis of six commercialized GM soybean varieties and related processed foods in Korea.     

A novel screening approach based on six real-time PCR systems for the detection of crustacean species in food

Analysis of crustacean species plays a role in authenticity issues as well as allergen detection. A real-time PCR-based screening assay was developed for the detection of crustaceans in food. In order to cover most relevant species in one analytical step, PCR systems were newly developed for the detection of shrimps (Penaeidae), lobster (Homarus sp.), Common shrimp (Crangon crangon), river prawns (Macrobrachium sp.) and Chinese mitten crab (Eriocheir sinensis). In addition a published system targeting Northern prawn (Pandalus borealis) was selected. All PCR-systems are based on mitochondrial 16S rRNA gene sequences and were optimized to be run with a standard program at a universal annealing temperature of 60 °C. Validation experiments confirmed a sensitivity of the PCR systems of 0.01–0.1 genome copies or 0.04–2.5 pg DNA, respectively. Specificity was demonstrated with 25,000 copies of pure genomic DNA from about thirty plant and animal species relevant in food and the performance in food matrix was evaluated. Finally primer and probes were pre-spotted steadily on 96-well PCR plates and the practical applicability of the assay was proven with selected food products. The assay enables detection of multiple species of market relevance.     

Event-specific analytical methods for six genetically modified maize events using visual and real-time loop-mediated isothermal amplification

Currently 138 genetically modified (GM) maize events have been authorized for commercial cultivation, comprising more than 65 per cent stacked events. With the increase in number of GM maize events globally, cost- and time-efficient diagnostics with on-site applicability are required to check for authorized GM events. Six GM maize events, namely, Bt11, GA21, MON810, MON89034, NK603 and TC1507, also present in 89 stacked events, are being widely commercialized in more than 17 countries. Visual and real-time loop-mediated isothermal amplification (LAMP) assays targeting these six GM maize events are being reported in the present study. Specificity of the developed LAMP assays was confirmed using fourteen commercialized GM maize events. Limit of detection of visual and real-time LAMP assays targeting Bt11, GA21, MON810, MON89034 and TC1507, was up to 0.01%, detecting 8 target copies, and for NK603 event-specific assays, was up to 0.1% detecting 73 target copies. Practical applicability of developed LAMP assays was verified using a set of five stacked GM maize events, namely, Bt11 × GA21, MON89034 × NK603, MON89034 × NK603 × TC1507, TC1507 × NK603 and TC1507 × MON810; and six powdered maize samples of proficiency testing. The reported LAMP assays can be efficiently employed for screening for presence of selected GM maize events in single or stacked form.     

Fat removal with hydrolyzed corn starch for real-time qPCR detection of Salmonella enterica in ground beef in 4.5 hours without enrichment

The rapid detection of low number of Salmonella in ground beef ideally requires an effective and economic molecular assay. The molecular analysis for the detection of Salmonella in ground beef by the polymerase chain reaction requires efficient methodology for extraction of targeted cells and effective removal of PCR inhibitors from the sample. The efficacy of hydrolyzed corn starch for the removal of fat along with the use of activated charcoal coated with milk proteins to remove PCR inhibitors were assessed. Salmonella enterica ser. Enteritidis was detected by real-time quantitative PCR at a level of 1 CFU/g of ground beef in 25 g samples containing 7%, 15% or 27% fat without enrichment. This study documents that partially hydrolyzed corn starch functions as effectively as beta-cyclodextrin for selective removal of fat.     

Rapid detection and quantification of zearalenone-producing Fusarium species by targeting the zearalenone synthase gene PKS4

This work is the first report on developing a method to detect and quantify the zearalenone-producing Fusarium species in foodstuff by real-time PCR assay with SYBR Green I. Based on the polyketide synthase gene (PKS4) sequences of four zearalenone-producing Fusarium strains, a specific primer set was designed and used to detect and quantify zearalenone-producing Fusarium in foodstuff. The system developed under study required only 100 mg infected maize flour for test, had ability to detect down to 10 copies of the target gene per reaction, and produced reliable quantitative data over three orders of magnitude. Compared with the conventional methods, it is more rapid, specific and sensitive to detect potential zearalenone contamination in food or feed production.     

Multiplex real-time PCR assays for simultaneous detection of maize MON810 and GA21 in food samples

This work describes a quantitative multiplex real-time PCR method optimized for the detection of maize MON810 and GA21. The use of specific primers and of labeled probes by real-time PCR allowed for the simultaneous detection and confirmation of amplicon identity and increased the reliability of the technique and the number of PCR applications to food analysis.Two different endogenous genes, Zein and Adh1, were evaluated for quantitative use as accountable for continuous development of maize traits. The quantification is based on a calibration standard curve obtained with the DNA extracted from Certified Reference Materials (CRMs). The limit of detection (LOD) and limit of quantification (LOQ) of the triplex assays developed was set at 3 and 36 copy numbers respectively.     

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.     

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.     

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.     

A survey on genetically modified maize in foods commercialised in Portugal

Maize, the second most important genetically modified (GM) crop, has the highest number of authorised GM events for food and feed in the EU. To provide consumer's information, labelling for food products containing more than 0.9% of GM material is demanded by the actual EU legislation. Analysis of foods is then essential to detect and quantify GM maize material and verify the compliance with labelling information. The aim of the present work was to assess the presence of GM maize in a range of processed foods commercialised in Portugal between 2007 and 2010. For this purpose, screening of GM material was carried out by qualitative PCR targeting the 35S promoter and the NOS terminator, followed by the specific detection of Bt11, MON810, Bt176, GA21, MON863, NK603, TC1507 (also known as DAS1507), DAS59122 and MIR604 events. The identified maize events were confirmed and quantified by real-time PCR with hydrolysis probes. The overall results of GMO screening were 30% for 35S promoter, 10% for NOS terminator and 25% for identified events. The most frequently detected events were MON810, TC1507 and NK603, with one sample containing GA21, while the other events were not detected in any of the analysed foods. The quantitative results suggest the need for a more severe control since 4% of the analysed foods contained more than the threshold for labelling and none of them declared the presence of GMO.     

Colorimetric ELISA based on glucose oxidase-regulated the color of acid–base indicator for sensitive detection of aflatoxin B1 in corn samples

This study described a sensitive direct competitive enzyme-linked immunosorbent assay (ELISA) for naked-eye detection of aflatoxin B₁ (AFB₁) by using glucose oxidase (GOx)-regulated bromocresol purple (BCP) color change. GOx was used as an alternative to horseradish peroxidase (HRP) for oxidization of glucose into hydrogen peroxide and gluconic acid. BCP, whose color is significantly sensitive to pH variation, was used as a signal output. Under optimal conditions, the developed method exhibited a considerably high sensitivity for AFB₁ detection with a cutoff limit of 100 pg/mL by the naked eye. The reliability of the developed colorimetric ELISA using naked-eye detection showed no false negative and false positive results among 70 AFB₁ spiked tests. Furthermore, the developed method showed a good linear range of 25–200 pg/mL for AFB₁ quantitative detection with a half-maximal inhibitory concentration at 66.72 pg/mL, which was approximately 10-fold lower than that of conventional HRP-based ELISA (IC₅₀ = 707 pg/mL). The recoveries from four kinds of AFB₁-spiked concentrations in corn extract solutions ranged within 80.56%–108.53%, with a coefficient of variation range of 1.69%–11.86%. These results exhibited good agreement with those of LC–MS/MS method indicating an acceptable accuracy and precision for AFB₁ quantitative detection in actual corn samples. In brief, this study was the first to use a GOx-mediated color change of BCP in immunoassay for naked-eye detection of AFB₁. This study also provided a new method for high-throughput screening detection of other small molecular chemicals using naked eye in resource-constrained countries.     

Adventitious presence of transgenic events in the maize supply chain in Peru: A case study

Cultivation and trade of transgenic or genetically modified organisms (GMO) and commodities has become widespread worldwide. In particular, production of transgenic crops has seen an accelerated growth along with a complex regulatory process. Current Peruvian legislation prohibits import of transgenic seeds and cultivation of transgenic crops in National territory but allows import of GMO-derived products and commodities. In addition, there is legislation that mandates the labeling of food products containing transgenic ingredients but the labeling threshold is still under discussion and the enforcement of this law is on hold. In this context, we evaluated adventitious presence of transgenic events in locally traded yellow maize using PCR- and immuno-based detection methods. Our results indicated that contamination during the distribution system of lots derived from non-transgenic maize was unavoidable and generally below 1.0% (w/w). Transgenic event MON810 was found in truck-loads of nationally grown maize. In general, frequencies of GMO-derived targets in whole-grain lots were 2.2% (GMO content ≥ 1%), 16.4% (GMO content ≤ 1%) and 81.3% (GMO content below our detection levels). When samples of de-germinated maize where evaluated, frequencies were 25.6% (GMO content > 0.9%), 65.1% (GMO content ≤ 0.9%) and 9.3% (GMO content below our detection levels). We believe this information will aid policy makers in establishing a suitable threshold for trade and product labeling as well as to conduct further investigation on other crops and scenarios.