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.     

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.     

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.     

Loop-mediated isothermal amplification targeting insect resistant and herbicide tolerant transgenes: Monitoring for GM contamination in supply chain

Efficient detection strategies for genetically modified (GM) crops are required to effectively address some of the biosafety and post-release monitoring issues, as global adoption of GM crops has been unprecedently increased. Herbicide tolerance and insecticide resistance are the major traits in commercialized GM food crops. Visual as well as real-time detection system based on loop-mediated isothermal amplification (LAMP) targeting lepidopteron insect resistant cry1Ac, cry2Ab2 and glyphosate tolerant cp4-epsps genes has been reported. Specificity of LAMP assays were confirmed using fourteen GM events of four crops, namely, corn (MON810, NK603, Bt11, Bt176, MON89034), cotton (MON531, MON15985, GFM-cry1A, Event1, MLS9124, MON1445, MON15985 × MON88913), eggplant (EE1) and soybean (GTS40-3-2). Real-time LAMP was found sensitive enough to detect as low as 2 copies for cry1Ac and 4 copies for cry2Ab2 and cp4-epsps within 35 min using a calibration curve. The limit of detection (LOD) of visual LAMP assays was down to 0.01% (4 copies of GM content) which is lower than conventional PCR (detecting 40–400 copies depending on target). LAMP assays are faster and more user-friendly than conventional PCR and could be efficiently utilized for monitoring of GM contamination in food and feed supply chain, with high specificity and sensitivity. The developed assays, when combined with a fast DNA extraction method, will facilitate on-site detection to check the GM status of a sample or product at ports of entry and in farmers' fields.     

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.     

Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA

Isogenic NK603 DNA was degraded by sonication or heat and quantified using A₂₆₀ and two fluorescent dye methods. Quantitative PCR (qPCR) experiments were conducted by amplifying an SSIIb-3 endogenous control and an NK603 transgene in untreated, sonicated, and heat treated samples. qPCR reactions on sonicated DNA samples, based on A₂₆₀ quantification, provided 0.125%, 1.14% and 2.15% NK603; while heat treated samples, provided results of 0.128%, 1.42%, and 2.73% NK603. qPCR reactions on sonicated DNA samples, based on the fluorescent dye method, provided results of 0.18%, 0.861% and 1.74% NK603; while heat treated DNA samples, provided results of 0.18%, 1.02%, and 2.16% NK603. The data suggested that fluorescent dye-based quantifications yielded more accurate determinations of the percent genetically engineered (GM) content at higher concentrations, most likely because fluorescent dye quantifications resulted in additional copies of template added into the qPCR. The data in this study suggested that neither fluorescent dye nor spectrophotometric methods of quantification on highly degraded DNA translated into concordant measurements of qPCR amplifiable DNA and accurate C_t values.     

Monitoring the occurrence of genetically modified soybean and maize in cultivated fields and along the transportation routes of the Incheon Port in South Korea

In South Korea, imported genetically modified (GM) soybean and maize have been approved for both human consumption and use in animal feed, but not for use in cultivation in fields. This study was conducted to survey the spread of GM soybean and maize in South Korea using multiplex-PCR analysis methods. Cultivated soybean, wild soybean, and maize leaf samples were collected from 26 major areas of soybean cultivation throughout eight provinces. Roadside areas near a major grain port in Incheon were also surveyed to investigate the escape and spread of GM seeds and plants. Amplification results showed that no GM soybean or maize was collected from cultivated fields. However, four GM maize plants were found in samples collected from the roadside near a grain transporting company at the Incheon Port. Based on PCR analysis using GM maize event-specific primers, it was suggested that a maize plant may be Mon810, while the other plants may be stacked events: Mon863 × Mon810 or Mon88017 × Mon810.     

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.     

Two detection methods of genetically modified maize and the state of its import into Japan

We detected genetically modified maize (GM-maize) with two different methods and examined the state of its import into Japan. A method using a cetyltrimethylammonium bromide buffer efficiently extracted DNA. Measurement of the extracted DNA at 260/230 nm and 260/280 nm indicated that the DNA was pure enough for polymerase chain reaction (PCR) amplification. Agarose gel electrophoresis of the PCR amplified products resolved a band at 329 bp for the zein gene and 437 and 522 bp for the introduced genes in Bt11, 619 bp in Event176, 194 bp in MON810 and 231 bp in LIBERTY maize. GM-maize was also detected by an enzyme-linked immunosorbent assay (ELISA) and there was no conflict in the results between the ELISA and the PCR method. However, the PCR method had advantages in distinguishing the lines of GM-maize. GM-maize was contained in the maize seeds imported into Japan between January and March in 2000 and MON810 counted for the largest percentage of the GM-maize.     

Qualitative and quantitative detection of genetically modified maize and soy in processed foods sold commercially in Brazil by PCR-based methods

Qualitative and quantitative polymerase-chain-reaction-based methods to detect genetically modified (gm) soy (RoundupReady^TM soy) and maize (Bt176 Maximizer maize; Bt11 maize, MON810 Yield Gard corn, T25 Liberty^R Link maize) were applied to processed foods sold commercially in Brazil. In total 100 foods containing maize and 100 foods containing soy were analysed in 2000 and again in 2001. In 2000, 13% of the soy containing products and 8% of those containing maize were shown to consist of material derived from the corresponding genetically modified organisms. In five samples of the products containing gm soy <4% and in eight samples >4% of the soy derived material was identified as coming from gm soybeans, whereas in the case of products containing gm maize five samples of the maize derived material were found to contain <4% and three samples >4% of gm maize. In 2001, gm soy was found in 21% of the soy containing foods, and gm maize was found in 9% of the maize containing products. Eight samples of the products containing gm soy were shown to contain <4% and 13 samples >4% of gm soy, the corresponding values for gm maize were five samples <4% and four samples >4%.     

Monitoring the occurrence of genetically modified maize at a grain receiving port and along transportation routes in the Republic of Korea

The cultivation area of genetically modified (GM) crops is increasing all over the world. Though no land in the Republic of Korea is currently used for the cultivation of GM crops, GM crop imports for food and foraging purposes are continuously increasing. This may promote the unintentional escape of GM crops. This study was conducted to investigate whether imported GM maize is released into our environment during the transportation of grain in the Republic of Korea. Based on PCR analysis, most of the maize grains in the forage products were GM, and about 50% of the grains were germinated. Monitoring was conducted in two major grain receiving ports, 15 feed manufacturing plants, and 14 livestock barns in five provinces of the Republic of Korea from July to September 2007. We found many spilled maize grains around open storage areas of ports and along truck transportation routes near feed manufacturing plants. Established maize plants were not found at or around Incheon port. However, we found 18 established maize plants at the Gunsan port, 15 of which were GM. We also found eight GM maize plants around four feed manufacturing plants and in two livestock barns. Based on the event-specific PCR analysis, three maize events (NK603, Mon810, and TC1507) were identified. Though several GM maize plants were found around the port and feed manufacturing plants, most of these facilities were located inside the industrial park and were far from cultivated fields, likely rendering the impact of these GM maize on the natural environments negligible. However, most of the livestock barns were close to cultivated areas. Moreover, maize plants were cultivated for food or feed near some livestock barns. This practice may facilitate gene flow from GM maize to non-GM maize plants. Therefore, continuous monitoring is necessary to detect the occurrence of GM maize, and appropriate action should be taken to prevent genetic admixture in our environment.     

Crop-specific GMO matrix-multiplex PCR: A cost-efficient screening strategy for genetically modified maize and cotton events approved globally

Crop-specific GMO matrices of 199 genetically modified (GM) events, comprising 143 GM maize events with 75 genetic elements and 56 cotton events with 45 genetic elements, were developed to screen globally approved GM maize and cotton events. As per the compiled information in the matrix, frequently present genetic elements were identified using GMOSeek algorithm: eight genetic elements ([P-35S] [r-act] [T-35S] [T-nos] [pinII] [pat] [aad-1] [cp4 epsps]) in maize and four ([P-35S] [T-nos] [pat] [nptII]) in cotton. Based on the cost-efficiency, feasibility of plexing and coverage of GM events, maize-specific tetraplex PCR, targeting Cauliflower Mosaic Virus 35S promoter (P-35S), Agrobacterium tumefaciens nos terminator (T-nos), Cauliflower Mosaic Virus 35S terminator (T-35S) and endogenous alcohol dehydrogenase (Adh1) gene, with limit of detection (LOD) up to 0.5% was developed. For screening of GM cotton events, pentaplex PCR, targeting P-35S, T-nos, neomycin phosphotransferase II (nptII), phosphinothricin acetyltransferase (pat) and endogenous stearoyl-ACP desaturase (Sad1) gene, with LOD up to 0.25% was developed. Practical applicability of multiplex PCR assays was confirmed with six maize samples of proficiency testing and eight spiked cotton samples. The reported tetraplex and pentaplex PCR assays could efficiently screen 94% of maize and 93% of cotton events approved globally. The developed GMO matrices in combination with multiplex PCR could facilitate checking the GM status of seed samples or food and feed products, and monitoring for presence of authorized GMOs in food and supply chain. This approach can be easily employed by low resource GM testing laboratories in the developing countries, as the multiplex PCR assays are easy to operate with less time and cost inputs. The GMO matrices being presented herein are based on the current information of approved GM events of maize and cotton, which can be further upgraded by including new approved GM events. As most of the newly approved GM events are stacked versions of already commercialized GM events, the developed multiplex PCR assays could also be employed to screen for their presence.     

Real-time and visual loop-mediated isothermal amplification: Efficient GMO screening targeting pat and pmi marker genes

Rapid and efficient screening assays based on loop-mediated isothermal amplification (LAMP) were developed, targeting two marker genes, namely, phosphinothricin-N-acetyltransferase (pat) and phosphomannose isomerase (pmi). These marker genes are being employed in more than 35% of GM events approved globally. Specificity of developed visual and real-time LAMP assays was confirmed using seven GM events of two crops, viz., maize (3272, 59122, Bt11, Bt176, MIR604, TC1507), and cotton (Widestrike™). In visual LAMP, positive amplification can be directly analyzed by the colorimetric change from orange to green, whereas real-time LAMP is based on the monitoring of fluorescence signals as amplification and annealing curves. Visual LAMP was found sensitive enough to detect up to 0.05% GM content for pat and 0.1% for pmi within 40 min. Real-time LAMP efficiently detected up to 0.01% GM content within 30 min. Practical applicability of developed assays was confirmed using proficiency test samples of maize. LAMP assays for pmi gene have been reported for the first time. Due to portability of systems, the developed LAMP assays when combined with a fast DNA extraction method could facilitate on-site GMO screening.     

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.     

GMO matrix: A cost-effective approach for screening unauthorized genetically modified events in India

Use of a pragmatic, affordable and reliable approach for screening and detection of a large number of genetically modified (GM) crops/events is the need of hour. A cost-effective matrix approach to check the GM status of food/feed products and for screening the presence of authorized and unauthorized GM events in India is being reported in the present study. A genetically modified organism (GMO) screening matrix, with the information on 106 genetic element targets for detection of 141 GM events of 21 crops, is being presented. These include commercially cultivated Bt cotton events and other GM events, under field trials during the past six years (2006–2012) in the country. The information on GM events, which were either indigenously developed or imported for research purposes, is also presented in brief. Ten most frequently present targets, viz., [P-35S] [T-nos] [Os-Msca1] [cry1Ab] [cry1Ac] [cry1C] [cry2Ab] [GA20 oxidase1] [nptII] [bar], were identified to screen these events using a GMOseek algorithm. This user-friendly screening tool is flexible for further updates with the new GM events and targets/elements. The data reported here related to the GM crops/events in India and the related GMO matrix are valuable tools to assist in the detection of accidental presence of unauthorized GM events in the food and supply chain globally, as well as in the context of the new labelling requirements for food commodities, as per the amendment to enforce GM food labelling from January 2013 in India. The reported GMO matrix approach would facilitate efficient, rapid and cost-effective preliminary screening by eliminating the need for development of specific testing methodologies for each GM event.     

Natural mycotoxin contamination of maize (Zea mays L.) in the South region of Brazil

The natural co-occurrence of fungal metabolites in maize samples from the South region of Brazil was studied using an LC-MS/MS based multi-mycotoxin method. All maize samples (n = 148) were contaminated with fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂). Aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁) were detected in 38 and 11 samples, respectively, while zearalenone (ZEN) and deoxynivalenol (DON), which were first regulated in 2014, were found in 110 and 71 samples, respectively. Apart from regulated mycotoxins, a broad range of non-regulated metabolites, from Aspergillus, Fusarium, Alternaria, Penicillium and other microbes, were also detected in maize sample. Fusarin C, a possible carcinogenic compound to humans, produced by Fusarium species and not addressed by Brazilian legislation, was detected in 54.2% of maize samples. All analysed maize samples were found to be contaminated by at least ten different metabolites, with the largest number of metabolites found in the same sample being 51.     

Monitoring of genetically modified soybean events in sausage products in South Korea

The use of genetically modified (GM) ingredients in various types of food products has increased worldwide. In this study, qualitative real-time PCR assays targeting five GM soybean events (RRS, MON89788, A2704-12, A5547-127, and MON87701) were performed to monitor their use in sausage samples. Thirty sausage samples containing soybean ingredients on their label were collected from Korean food markets and analyzed. The endogenous lectin gene of soybean was used as an internal positive control. Real-time PCR showed that the threshold cycle (Ct) values ranged from 28.62 to 33.90 for the 30 sausage samples. Of the 30 sausage samples, 19 samples were negative for the presence of GM soybeans. In the positive samples, the results revealed three GM soybean events [Roundup Ready Soybean (RRS), A2704-12, and/or MON89788] in 11 of the 30 food samples tested. These results demonstrate that sausage products contain different GM soybean events.     

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.     

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.     

Inactivation of Aspergillus flavus spores by curcumin-mediated photosensitization

Minimizing fungal infection is essential to the control of mycotoxin contamination of foods and feeds but many potential control methods are not without their own safety concerns for the consumers. Photodynamic inactivation is a novel light-based approach which offers a promising alternative to conventional methods for the control of mycotoxigenic fungi. This study describes the use of curcumin to inactivate spores of Aspergillus flavus, one of the major aflatoxin producing fungi in foods and feeds. Curcumin is a natural polyphenolic compound from the spice turmeric (Curcuma longa). In this study the plant has shown to be an effective photosensitiser when combined with visible light (420 nm). The experiment was conducted in in vitro and in vivo where A. flavus spores were treated with different photosensitiser concentration and light dose both in buffer solution and on maize kernels. Comparison of fungal load from treated and untreated samples was determined, and reductions of fungal spore counts of up to 3 log CFU ml⁻¹ in suspension and 2 log CFU g⁻¹ in maize kernels were obtained using optimal dye concentrations and light dose combinations. The results in this study indicate that curcumin-mediated photosensitization is a potentially effective method to decontaminate A. flavus spores in foods and feeds.