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.     

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.     

Monitoring of Roundup™ Ready soybean in Guangdong province in China

This study was aimed at monitoring the presence of Roundup™ Ready soybean (RRS) in the market (660 samples) and fields (630 samples) in Guangdong Province in China using polymerase chain reaction (PCR) method. According to the gene sequence transgened to Roundup Ready™ soybean, four primer pairs, aiming at cp4-epsps, lectin, CaMV 35S promoter and nos terminator were designed and PCR method to detect the GM soybean has been established. The detection results demonstrate the presence of one Roundup™ Ready soybean seed sample from 60 samples in Shenzhen City. No soybean plant samples from Guangdong Province were found.     

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.     

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.     

Real-time PCR-based detection and quantification of genetically modified maize in processed feeds commercialised in Malaysia

The present study which dealt mainly with processed feeds and some maize samples sold commercially in Malaysia evaluated the implementation of a real-time PCR cycling system for singleplex screening of eight target sequences (lectin, hmg, adh1, p35S, NK603, GA21, MON810 and MON863) and quantification of four genetically modified (GM) maize events (NK603, GA21, MON810 and MON863). The effects of using proprietary glass magnetic particles to bind DNA to their surface were also investigated in terms of DNA quantity, purity, integrity, quality and its overall effect on DNA amplification. GM material was present in 26.2% feeds and 65% maize samples. All GM samples contained MON810 followed by NK603 (47.5%), GA21 (25%) and MON863 (2.5%). Single-event and multiple-events were identified in the GM samples with 50% of the GM samples containing multiple-events. The present study which represents a fast and reliable methodology would provide an overview of the presence and levels of GMOs in feeds and maize in Malaysia.     

Detection of eight genetically modified canola events using two event-specific pentaplex PCR systems

An event-specific multiplex polymerase chain reaction (PCR) detection method was developed to simultaneously detect eight genetically modified (GM) canola events (GT73, MS1, MS8, RF1, RF2, RF3, T45, and Topas 19/2). For a successful multiplex PCR assay, the eight GM canola events were divided into groups 1 and 2 in consideration of their amplicon sizes, primer efficiencies, and target sequences. In addition to the canola endogenous reference gene, FatA, the two pentaplex PCR assays targeted group 1, containing GT73, MS8, RF3, and T45, and group 2, including MS1, RF1, RF2, and Topas 19/2. Event-specific primers targeting the eight GM canola events were designed, and their specificities were confirmed using 14 GM events of maize, soybean, cotton, and canola. After optimizing the reaction conditions, the limits of detection of these two assays were approximately 0.025% for group 1 and 0.0125% for group 2. This multiplex PCR method for eight GM canola events was validated by two operators, and the data confirmed the reliability of the developed assays. The method was used to test commercially available canola seed (eight samples) and meal (one sample) produced in South Korea, China, Canada, and Australia, and the results revealed one or more GM canola events in seven of the nine samples tested. These results show that the developed multiplex system is applicable for use in the specific testing of eight commercially available GM canola events.     

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.     

Comprehensive matrices for regulatory approvals and genetic characterization of genetically modified organisms

The production of new types of genetically modified organisms (GMOs) and the use of products containing or derived from these materials are expanding globally. This poses a challenge in providing cost-effective comprehensive analyses. In this line, the state of art testing approaches rely on a matrix representing the GM events with their corresponding GM markers - DNA elements used in plants' transformation. Accordingly, this study aimed first at constructing an updated and comprehensive matrix of genetic characterization of GM events based on an extensive review of the relevant databases. Inclusive lists of 356 GM markers and 508 events in 29 plant species were compiled and organized into a matrix. The frequency of occurrence of these elements was then determined. Moreover, for the first time, a matrix representing the regulatory status of every compiled GM event was established. Remarkably, numerous inconsistencies were detected among the databases at the levels of nomenclature, events' registry, molecular characterization and regulatory approvals. Both matrices represent a useful tool for comprehensive and cost-effective analyses. The genetic matrix permits designing the most straightforward testing strategy that provides the maximum information about GMOs in a sample in the minimum number of experimental steps. Moreover, the novel regulatory matrix, allows further decreasing the number of required event-specific identification tests by giving higher probabilities to those authorized in the samples' country of origin. Finally, the genetics and regulatory matrices represent the building-block for establishing an inclusive automated database for GMOs which is instrumental for testing laboratories worldwide.     

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.     

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 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.     

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.     

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.     

A multiplex-conventional PCR assay for bovine,ovine, caprine and fish species identification in feedstuffs: Highly sensitive and specific

A multiplex PCR assay was developed for rapid and reliable identification of bovine, ovine, caprine and fish species in feedstuffs simultaneously. The method merges the use of bovine, ovine, caprine and fish primers that amplify fragments (ovine; 119 bp, caprine; 142 bp, fish; 224 bp and bovine; 271 bp) of the mitochondrial t.glu gene forward and cyt b reverse, 12S rRNA, 12S rRNA, and ATPase subunit 8 genes respectively, and a universal 18S rRNA primers that amplifies a 99 bp from eukaryotic DNA. To evaluate the effect of heat treatment, a severe sterilization condition (133 °C at 300 kPa for 20 min) was applied. Multiplex analysis of the reference feedstuff samples showed that the detection limit of the assay was 0.01% for each species. Taken together, all data indicated that this multiplex PCR assay was a simple, rapid, sensitive, specific, and cost-effective detection method for bovine, ovine, caprine and fish species in feedstuffs.     

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.     

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.     

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.