Yanfang Yuan

Safety assessment of Cry1Ab/Ac fusion protein.

Cry1ab/ac gene was fused by both the cry1ab gene (GenBank Accession No. X54939) and the cry1ac gene (GenBank Accession No. Y09787), which was widely used in genetically modified (GM) rice, cotton, maize and so on. In order to support the safety assessment of GM food or feed products containing Cry1Ab/Ac protein, sufficient quantities of Cry1Ab/Ac protein were produced in Escherichia coli for in vitro evaluation and animal studies. The Cry1Ab/Ac protein does not possess the characteristics associated with food toxins or allergens, i.e., it has no sequence homology with any known allergens or toxins, and no N-glycosylation sites, can be rapidly degraded in gastric and intestinal fluids, and is devoid of adverse effects in mice by gavage at a high dose level of 5g (Cry1Ab/Ac protein)/kg body weight. In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the Cry1Ab/Ac protein in human food or animal feed.

A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on.

Universal Primer‐Multiplex PCR Approach for Simultaneous Detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in Food Samples

Escherichia coli, Listeria monocytogenes, and Salmonella spp. are 3 kinds of the most important food-borne human pathogens. Traditional microbiological analysis is labor-intensive, time-consuming, and easily contaminated, thus producing false positive signals; it also involves much subjectivity judgments. Multiplex-PCR could be applied to detect multiple target organisms simultaneously to save time and labor, but there is always disproportionate amplification resulting from the disparity of different primers. To gain a rapid and sensitive method, a universal primer-multiplex PCR system (UP-M-PCR) was developed and applied for simultaneous detection of the 3 organisms. This method simplified traditional multiplex-PCR reaction system and overcame its amplification disparities among different primers; moreover, it got a high specificity and sensitivity (85, 155, and 104 copies/reaction for E. coli O157, L. monocytogenes, and Salmonella spp., respectively). Compared with the time-consuming and laborious microbiological analysis, UP-M-PCR had a lower risk of cross-contamination without inoculation and incubation. Test results for 36 food samples showed that UP-M-PCR method got a relative accuracy of 91.77% when compared with traditional microbiological analysis. It could serve as a rapid screening method for pathogen detection and could detect target genes even in dead pathogenic cells. In addition, it has the potential to be performed in an automation mode and might find broader application in simultaneous detection of other multiple pathogens.

Event-Specific Detection of Stacked Genetically Modified Maize Bt11 × GA21 by UP-M-PCR and Real-Time PCR

More and more stacked GMOs have been developed for more improved functional properties and/or a stronger intended characteristic, such as antipest, improved product efficiency etc. Bt11 x GA21 is a new kind of stacked GM maize developed by Monsanto Company. Since there are no unique flanking sequences in stacked GMOs, up to now, no appropriate method has been reported to accurately detect them. In this passage, a novel universal primer multiplex PCR (UP-M-PCR) was developed and applied as a rapid screening method for the simultaneous detection of five target sequences (NOS, 35S, Bt11 event, GA21 event, and IVR) in maize Bt11 x GA21. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers. Whats more, it got a high specificity and had a detection limit of 0.1% (approximates to 38 haploid genome copies). Furthermore, real-time PCR combined with multivariate statistical analysis was used for accurate quantification of stacked GM maize Bt11 x GA21 in 100% GM maize mixture (Bt11 x GA21, Bt11 and GA21). Detection results showed that this method could accurately validate the content of Bt11, GA21 and Bt11 x GA21 in 100% GM mixture with a detection limit of 0.5% (approximates to 200 haploid genome copies) and a low relative standard deviation <5%. All the data proved that this method may be widely applied in event-specific detection of other stacked GMOs in GM-mixture.

Safety assessment of Cry1C protein from genetically modified rice according to the national standards of PR China for a new food resource

The Cry1C protein produced in Escherichia coli was used for in vitro evaluation and animal studies to support the safety assessment of GM food or feed products containing the Cry1C protein. The Cry1C protein does not have any sequence homology with known allergens or toxins. Although the Cry1C protein was heat stable it was rapidly degraded in vitro with simulated gastric or intestinal fluids. It did not cause adverse effects in mice as administered by gavage at a high level dosage of 5 g (Cry1C protein)/kg body weight. The mutagenicity of this protein was evaluated according to the national standards of Peoples Republic of China (PR China) for a new food resource. In mutagenic tests, the Cry1C protein caused<4 micronucleated cells per 1000 cells, <16 sperm abnormalities per 1000 cells and was not associated with any increased mutations in the Ames test. Taken together, these data indicate that the Cry1C protein is not a potential allergen or toxin.

Survey of acrylamide levels in Chinese foods

A survey of levels of acrylamide (AA) in 349 food products obtained from the Chinese market was conducted. AA was determined by an liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The limit of detection (LOD) and the limit of quantification (LOQ) of the method in four different matrices ranged from 0.8 to 10 µg kg−1 and from 4.0 to 25 µg kg−1, respectively. The results from this survey indicated that AA was present in all samples except drinking water and tea. AA contents in different samples varied greatly according to the raw materials and processing conditions. The highest level of AA was found in potato products, with an average level of 1467 µg kg−1. Preliminary estimates of AA exposure and risk assessment of AA from foods in the Chinese population were performed using a combination of data obtained in the present survey and 2002 dietary consumption survey carried out in 2002 for the Chinese population. The average dietary exposure of AA was estimated to be 0.38 µg kg−1 body weight day−1, which is relatively low compared with the result reported by the Food and Agricultural Organization/World Health Organization (FAO/WHO). Furthermore, the margin of exposure for neurotoxicity, reproductive toxicity, and carcinogenicity of AA was calculated to be 1318, 5250, and 787, respectively.

Safety Assessment of Transgenic Bacillus thuringiensis Rice T1c-19 in Sprague-Dawley Rats from Metabonomics and Bacterial Profile Perspectives

Bacillus thuringiensis rice is facing commercialization as the main food source in the near future. The unintended effects of genetically modified (GM) organisms are the most important barriers to their promotion. We aimed to establish a new in vivo evaluation model for genetically modified foods by using metabonomics and bacterial profile approaches. T1c‐19 rice flour or its transgenic parent MH63 was used at 70% wt/wt to produce diets that were fed to rats for ∼ 90 days. Urine metabolite changes were detected using 1H NMR. Denaturing gradient gel electrophoresis and real‐time polymerase chain reaction (RT‐PCR) were used to detect the bacterial profiles between the two groups. The metabonomics was analyzed for metabolite changes in rat urine, when compared with the non‐GM rice group, where rats were fed a GM rice diet. Several metabolites correlated with rat age and sex but not with GM rice diet. Significant biological differences were not identified between the GM rice diet and the non‐GM rice diet. The bacteria related to rat urine metabolites were also discussed. The results from metabonomics and bacterial profile analyses were comparable with the results attained using the traditional method. Because metabonomics and bacterial profiling offer noninvasive, dynamic approaches for monitoring food safety, they provide a novel process for assessing the safety of GM foods.

Effects of genetically modified T2A-1 rice on faecal microflora of rats during 90 day supplementation.

BACKGROUND Many animal studies have been performed on products with the Bacillus thuringiensis insecticidal toxin-encoding gene (Bt products), but less have focused on its effects on intestinal microflora owing to difficulties in culturing. This 90 day study was designed to assess unintended effects of genetically modified T2A-1 rice (GMR) on selected intestinal bacteria (Lactobacillus group, Bifidobacterium genus, Escherichia coli subgroup, Enterococcus genus and Clostridium perfringens) of rats by the real-time polymerase chain reaction (PCR) method. RESULTS During the whole experiment, no statistically significant differences in the numbers of specific bacteria and total bacteria were found between the GMR group and its parental group. At all stages of the experiment the two main probiotics (Lactobacillus group and Bifidobacterium genus) in faeces accounted for 11-23% of the total bacteria, whereas the conditional pathogens (E. coli subgroup, Enterococcus genus and C. perfringens) made up less than 1% of the total bacteria. B/E (log(10) copies of Bifidobacterium genome g(-1) faeces/log(10) copies of E. coli genome g(-1) faeces) ratios from 1.19 to 1.34 were obtained. Furthermore, significant correlations (P < 0.01) between the real-time PCR method and the plate count method were found, with r values ranging from 0.60 to 0.75. CONCLUSION No adverse effects on the numbers of specific bacteria in rat faeces were observed as a result of GMR feeding. The real-time PCR method is recommended in further studies on the composition and dynamics of the intestinal bacteria community for better safety assessment of GM materials.

Unintended effects were investigated in antioxidant activity between genetically modified organisms and their nontransgenic control

Other than the targeted approach on compositional analysis, non-targeted approaches on genomics, proteomics and metabolomics are developing to search for unintended effects with respect to genetically modified (GM) food safety assessments. Antioxidant activity system was closely related with plant growth and reproduction as well as human health. This study was to investigate some other potential unintended effects from a range of primary and secondary metabolites by comparison of antioxidant activity system between six pairs of GMOs and their nontransgenic control. Antioxidant activity system was explored in total phenolics, unsaturated fatty acids and oxido-reductase activity analysis (including peroxidase (POD), polyphenol oxidase (PPO), catalase activity (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR)). The results from oxido-reductase activity analysis indicated significant differences (P < 0.05) between GMOs and their nontransgenic control, except for a few enzymatic activities of several GM crops. The data of total phenolics and unsaturated fatty acids also showed significant differences (P < 0.05) between GMOs and their nontransgenic control. However, no obvious differences occurred among all tested maize samples or canola samples. Key words: Unintended effects, antioxidant activity, genetically modified organisms, maize, canola.

The effect of genetically modified Lactobacillus plantarum 590 on the gut health of sprague–dawley rats

Lp was a generally recognized as safe microorganism. Lactobacillus plantarum 590 was obtained by inserting nisI gene into Lp genome to help it tolerate higher concentration nisin. As the unintended effects of the genetically modified microorganism (GMM) are the most important barriers to the progress of GMM, we have performed a useful exploration to establish a new in vivo evaluation model for GMM from the point of view of intestinal health. In this study, Sprague–Dawley rats were orally administered with Lp 590 and Lp for 4 weeks. Fecal samples were collected to determine the number of beneficial bacteria Bifidobacterium and harmful bacteria Clostridium perfringens. Denaturing gradient gel electrophoresis was used to detect the bacterial profiles of every group. Fecal enzyme activities and short‐chain fatty acids as main metabolites were also examined. Real time PCR (RT‐PCR) and immunohistochemistry were used to analyze two proteins (ZO‐1 and occludin) and secretory immunoglobulin A to detect intestinal permeability and mucosal immunity, gut permeability and gut mucosal immunity were analyzed to see whether GM Lp 590 can induce changes of the gut health when compared with non‐GM Lp group, andeventually we concluded that there is no significant difference between GM Lp 590‐fed group and non‐GM Lp‐fed group. The conclusion of gut health test was comparable withthat from traditional subchronic test. Evaluation of intestinal health will be a new approach of assessing the safety of GMM.