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DNA analysis of processed foods is performed widely to detect various targets, such as genetically modified organisms (GMOs). Food processing often causes DNA fragmentation, which consequently affects the results of PCR analysis. In order to assess the effects of DNA fragmentation on the reliability of PCR analysis, we investigated a novel methodology to quantify the degree of DNA fragmentation. We designed four real-time PCR assays that amplified 18S ribosomal RNA gene sequences common to various plants at lengths of approximately 100, 200, 400, and 800 base pairs (bp). Then, we created an indicator value, "DNA fragmentation index (DFI)", which is calculated from the Cq values derived from the real-time PCR assays. Finally, we demonstrated the efficacy of this method for the quality control of GMO detection in processed foods by evaluating the relationship between the DFI and the limit of detection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize event, MIR162. We first prepared a standard plasmid for MIR162 quantification. The conversion factor (Cf) required to calculate the genetically modified organism (GMO) amount was empirically determined for two real-time PCR instruments, the Applied Biosystems 7900HT (ABI7900) and the Applied Biosystems 7500 (ABI7500) for which the determined Cf values were 0.697 and 0.635, respectively. To validate the developed method, a blind test was carried out in an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr). The determined biases were less than 25% and the RSDr values were less than 20% at all evaluated concentrations. These results suggested that the limit of quantitation of the method was 0.5%, and that the developed method would thus be suitable for practical analyses for the detection and quantification of MIR162.

Genetically modified (GM) papaya (Carica papaya L.) line 55-1 (55-1), which is resistant to papaya ringspot virus infection, has been marketed internationally. Many countries have mandatory labeling regulations for GM foods, and there is a need for specific methods for detecting 55-1. Here, an event- and construct-specific real-time polymerase chain reaction (PCR) method was developed for detecting 55-1 in papaya products. Quantitative detection was possible for fresh papaya fruit up to dilutions of 0.001% and 0.01% (weight per weight [w/w]) for homozygous SunUp and heterozygous Rainbow cultivars, respectively, in non-GM papaya. The limit of detection and quantification was as low as 250 copies of the haploid genome according to a standard reference plasmid. The method was applicable to qualitative detection of 55-1 in eight types of processed products (canned papaya, pickled papaya, dried fruit, papaya-leaf tea, jam, puree, juice, and frozen dessert) containing papaya as a main ingredient., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this article, we report a novel real-time PCR-based analytical method for quantitation of the GM maize event LY038. We designed LY038-specific and maize endogenous reference DNA-specific PCR amplifications. After confirming the specificity and linearity of the LY038-specific PCR amplification, we determined the conversion factor required to calculate the weight-based content of GM organism (GMO) in a multilaboratory evaluation. Finally, in order to validate the developed method, an interlaboratory collaborative trial according to the internationally harmonized guidelines was performed with blind DNA samples containing LY038 at the mixing levels of 0, 0.5, 1.0, 5.0 and 10.0%. The precision of the method was evaluated as the RSD of reproducibility (RSDR), and the values obtained were all less than 25%. The limit of quantitation of the method was judged to be 0.5% based on the definition of ISO 24276 guideline. The results from the collaborative trial suggested that the developed quantitative method would be suitable for practical testing of LY038 maize.

DNA was extracted from sweet corn and its processed products using four DNA extraction methods: the CTAB method, the DNeasy Plant Maxi kit, GM Quicker 3, and Genomic-tip 20/G. DNA was successfully extracted from raw sweet corn and baby corn samples using all four methods. Meanwhile, from frozen, canned, and dry pack products, DNA was well extracted using the DNeasy Plant Maxi kit, GM Quicker 3, and Genomic-tip 20/G, but not enough with the CTAB method. The highest yield of DNA was obtained with Genomic-tip 20/G. The degree of degradation of extracted DNA was observed to increase in the order of raw, frozen, canned, dry pack, and baby corn samples. To evaluate the quality of extracted DNA, real-time PCR analyses were conducted using three maize endogenous genes. The DNAs extracted using GM Quicker 3 had high purity, suggesting that GM Quicker 3 would be the most suitable method for DNA extraction from processed sweet corn products.

A GM maize event, MIR604, has been widely distributed and an analytical method to quantify its content is required to monitor the validity of food labeling. Here we report a novel real-time PCR-based quantitation method for MIR604 maize. We developed real-time PCR assays specific for MIR604 using event-specific primers designed by the trait developer, and for maize endogenous starch synthase IIb gene (SSIIb). Then, we determined the conversion factor, which is required to calculate the weight-based GM maize content from the copy number ratio of MIR604-specific DNA to the endogenous reference DNA. Finally, to validate the developed method, an interlaboratory collaborative trial according to the internationally harmonized guidelines was performed with blind samples containing MIR604 at the mixing levels of 0, 0.5, 1.0, 5.0 and 10.0%. The reproducibility (RSDr) of the developed method was evaluated to be less than 25%. The limit of quantitation of the method was estimated to be 0.5% based on the ISO 24276 guideline. These results suggested that the developed method would be suitable for practical quantitative analyses of MIR604 maize.

We investigated the GM maize grain content of non-identity preserved (non-IP) maize samples produced in 2009 in the USA using our individual kernel detection system, involving two multiplex qualitative PCR methods coupled to microchip electrophoresis and partially real-time PCR array analysis, to clarify how many GM event maize grains were present in the samples and which GM events frequently appeared in 2009. The average percentage and standard deviation of GM maize grains on a kernel basis in five non-IP sample lots were 81.9%±2.8%, the average percentage of single GM event grains was 46.9%, and the average percentage of stacked GM event grains was 35.0%. MON88017 grains and NK603 grains were the most frequently observed as single GM event grains. The most frequent stacked GM event grains were MON88017×MON810 grains. This study shows that our method can provide information about GM maize events present in imported maize samples on a kernel basis.

Developments in the use of genetically modified plants for human and livestock health and phytoremediation were surveyed using information retrieved from Entrez PubMed, Chemical Abstracts Service, Google, congress abstracts and proceedings of related scientific societies, scientific journals, etc. Information obtained was classified into 8 categories according to the research objective and the usage of the transgenic plants as 1: nutraceuticals (functional foods), 2: oral vaccines, 3: edible curatives, 4: vaccine antigens, 5: therapeutic antibodies, 6: curatives, 7: diagnostic agents and reagents, and 8: phytoremediation. In total, 405 cases were collected from 2006 to 2010. The numbers of cases were 120 for nutraceuticals, 65 for oral vaccines, 25 for edible curatives, 36 for vaccine antigens, 36 for therapeutic antibodies, 76 for curatives, 15 for diagnostic agents and reagents, and 40 for phytoremediation (sum of each cases was 413 because some reports were related to several categories). Nutraceuticals, oral vaccines and curatives were predominant. The most frequently used edible crop was rice (51 cases), and tomato (28 cases), lettuce (22 cases), potato (18 cases), corn (15 cases) followed.

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.

Genetically modified (GM) foods must be tested for safety, including by allergenicity tests to ensure that they do not contain new allergens or higher concentrations of known allergens than the same non-GM foods. In this study experimentally developed EGFP-transgenic chickens were used and evaluated the allergenicity of meat from the chicken based on a serological and two-dimensional difference gel electrophoresis (2D-DIGE) analysis. For the serological analysis, a Western blotting with allergen-specific antibodies and a proteomic analysis of chicken meat allergens with patients' sera, a so-called allergenome analysis, were used. The allergenome analysis allowed us to identify five IgE-binding proteins in chicken meat, including a known allergen, chicken serum albumin, and no qualitative difference in their expressions between the GM and non-GM chicken meat was found. Results of the 2D-DIGE analysis showed that none of the IgE-binding proteins in chicken meat were significantly changed in expression levels between non-GM and GM chicken, and only 3 of the 1500 soluble protein spots including green fluorescence protein were markedly different as a result of gene transfer. These above results showed that the combination of serological and 2D-DIGE analysis is a valid method of evaluating quality and quantity of allergens in GM foods., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

A novel real-time PCR-based analytical method was established for the event-specific quantification of a GM soybean event MON89788. The conversion factor (C(f)) which is required to calculate the GMO amount was experimentally determined. The quantitative method was evaluated by a single-laboratory analysis and a blind test in a multi-laboratory trial. The limit of quantitation for the method was estimated to be 0.1% or lower. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were both less than 20%. These results suggest that the established method would be suitable for practical detection and quantification of MON89788.

A real-time polymerase chain reaction (PCR) method specific for genetically modified (GM) maize event DAS59132 (E32) was adapted for qualitative detection of low level presence of E32. The method was validated by a collaborative trial with eight participating Japanese laboratories. Sensitivity was assessed with three different samples of corn flour fortified to 0%, 0.05% and 0.1% (w/w) E32 respectively. In addition, a 0.01% E32 DNA solution was used. The detection limit with DNA solution was estimated to be approximately 0.01%. In conclusion, the results of the study confirmed this real-time PCR method as a reliable tool for qualitative detection of E32 maize.

The Bt11 maize-specific qualitative detection method based on polymerase chain reaction (PCR) in the JAS analytical test handbook has been widely used for administrative monitoring of GM crops and quality control of commercially distributed grains. In the present investigation, some apparently false-positive detections were observed in assays using the Bt11 maize-specific method, and these erroneous results were proved to have been caused by non-specific DNA amplification. We improved the detection method to reduce non-specific amplification by decreasing the concentration of magnesium ions in the PCR mixture. The subsequent evaluation of analytical performance demonstrated no marked difference between the currently used and the improved methods, except for the reduced non-specific amplification. We conclude that the currently used standard method should be replaced with the improved method for the reliable detection of Bt11 maize.

Genetically modified (GM) foods are beneficial from the standpoint of ensuring a constant supply of foodstuffs, but they must be tested for safety before being released on the market, including by allergenicity tests to ensure that they do not contain new allergens or higher concentrations of known allergens than the same non-GM foods. In this study we used GM-amago salmon into which a growth hormone gene had been introduced and compared the allergens contained in the GM and the non-GM-amago salmons. We used a combination of Western blotting with allergen-specific antibodies and a proteomic analysis of their allergens with patients' sera, a so-called allergenome analysis, to analyze allergens. Western blotting with specific antibodies showed no increase in the content of the known allergens fish parvalbumin and fish type-I collagen in GM-amago salmon, in comparison with their content in non-GM-amago salmon. The allergenome analysis of two fish-allergic patients allowed us to identify several IgE-binding proteins in amago salmon, including parvalbumin, triose-phosphate isomerase, fructose-bisphosphate aldolase A, and serum albumin, and there were no qualitative differences in these proteins between GM and non-GM-amago salmons. These results indicate that amago salmon endogenous allergen expression does not seem to be altered by genetic modification.

A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this.

A new real-time PCR method using capturing oligo-immobilized PCR tubes is described. This method was used to detect specific genes for soybean and genetically modified (GM) soybean in food matrices. In a standard reaction using soybean genomic DNA and a capturing oligo for the lectin gene (Le1) immobilized on the tube, we examined the effects of such hybridization conditions as the location, length, and amount of the capturing oligo, and the incubation time and temperature. Under optimized conditions, the copy number of Le1 was determined in a concentration-dependent manner from soybean genomic DNA and soybean lysate (DNA 10-1000 ng, r=0.99; lysate 1-100%, r=0.99). The copy number of a Roundup Ready soybean (RRS) gene was also successfully detected in a concentration-dependent manner (1-100%, r=0.99) from GM soybean lysate, using PCR tubes with an immobilized capturing oligo for the transgene. Our data indicate that this is a rapid and simple method to determine specific genes for soybean and GM soybean in food matrices.

Enzyme-linked immunosorbent assay (ELISA) is the most convenient method of monitoring the occurrence of IgE antibodies specific for novel proteins in genetically modified (GM) foods. The levels of IgE specific for a recombinant protein, Cry1Ab, were determined using an ELISA method. A soluble form of the Cry1Ab protein purified from pCold1 vector-transformed Escherichia coli pTf16/BL21 was used as the ELISA coating antigen, and 1M NaCl was used as the washing buffer to remove IgE non-specifically bound to the coated antigen. Sera from 44 patients allergic to major food allergens were obtained, diluted 20-fold, tested, and found no identifiable IgE above background levels. We also tested sera from patients with corn allergy against whole extracts of non-GM and GM-corn (MON 810) using immunoblotting. The staining patterns were similar for the two types of corn. These results indicate that significant levels of IgE antibodies specific to Cry1Ab were not found in the sera of Japanese patients with food allergies.

We examined the lateral flow strip assay for identifying unauthorized genetically modified (GM) rice. The GM rice expresses the Bacillus thuringiensis (Bt) toxin, CryIAc protein, which confers tolerance to insects. The recombinant CryIAc protein was prepared from the inclusion bodies of an E. coli. strain into which the CryIAc gene had been inserted, using gel filtration chromatography. The lateral flow strip assay for the identification of GM cotton which also expresses CryIAc protein, was applied to unpolished rice and polished rice spiked with recombinant CryIAc protein. The spiked recombinant CryIAc protein was clearly detected at the level of 0.012 microg/g in both the unpolished and polished rice. After loading of the extract on the strip, a 60 -minute stand time is necessary to clearly detect CryIAc protein. The detection limit was approximately 12 ng CryIAc protein per gram of rice. These results suggest that the lateral flow strip assay for GM cotton can be used to detect CryIAc protein expressed in GM rice.

Subchronic animal feeding studies to examine the effect on the immune system of genetically modified corn CBH351, which contains the Cry9C protein derived from Bacillus thuringiensis subspecies tolworthi, were conducted in female BN rats and B10A mice. The studies were designed to compare the effect of a line of genetically modified corn CBH351 (GM corn) with that of isoline corn (non-GM corn). Heat-treated corn meal was incorporated into the diets of the rats and mice at a concentration of 50%. The study duration was 13 weeks. Growth, food intake, and organ weights of the thymus, spleen, and liver were compared between animals fed the non-GM and GM lines. The histological findings in thymus, spleen, mesenteric lymph nodes, Peyer's patches, small intestines, liver, kidney, and bone marrow, and the presence of Cry9C-specific IgE, IgG, IgG1 and IgA antibodies in serum were also compared. The results showed no significant differences in growth, feeding value, or the histological findings in immunity-related organs between the animals fed the GM and non-GM lines. Production of Cry9 C-specific IgE and IgA was not detected in the serum of either group. Production of Cry9C-specific IgG and IgG1 was slightly increased in the 50% GM groups of BN rats. No Cry9C-specific IgG or IgG1 was detected in the serum of BN rats fed the diet containing 5% GM-corn In conclusion, no immunotoxic activity was detected in the GM-corn-fed rats and mice in this subchronic dietary study.

We performed experiments on in vitro digestion of newly expressed proteins by SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) to assess the allergenicity of food components derived from biotechnological modification. For newly expressed proteins, we chose CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4) and Cry1Ab derived from Bacillus thuringiensis subsp. kurstaki strain HD-1. The former is expressed in GM-soybeans and the latter is expressed in GM-corns. Firstly, we examined the digestibility of purified CP4-EPSPS and Cry1Ab by SGF. Both proteins were rapidly digested within 60 sec. After preheating, the digestibility by SGF was slightly increased. Secondly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SGF. The digestion time of both proteins by SGF was almost the same as that of the purified proteins. Thirdly, the digestibility of CP4-EPSPS and Cry1Ab by SIF was examined. The digestion time of these proteins was 240 min or more. However, digestibility of these proteins by SIF was dramatically increased by preheating, and the digestion time was less than 5 sec. Fourthly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SIF. Digestion time of both proteins by SIF was almost the same as that of the purified proteins. From these results, we concluded that the digestibility of both CP4-EPSPS and Cry1Ab by SGF and SIF was increased by preheating. Therefore, we suggest that the allergenicity of both proteins should be extremely low because of the easy digestibility of these proteins by SGF and also by SIF with preheating.