加工产品中转基因玉米Bt11成分实时荧光PCR定量(性)检测

实验在玉米自身基因和外源基因的边界序列之间设计了具有品种和品系特异性的引物和探针 ,并以实时荧光PCR技术 ,建立了加工产品中转基因玉米Bt1 1成分品系鉴定检测和定量检测的方法。实验对加热条件和时间对检测转基因成分的影响作了探讨 ,并检测了部分市售食品和饲料。检测结果发现 ,加热时间温度越高、时间越长 ,对转基因成分定量检测的影响越大 ;在所检测的样品中可以检测出转基因玉米Bt1 1成分 ,有些样品还同时检出其他转基因成分。本研究实验建立的方法 ,可以用于加工产品中转基因成分的定量检测 ,也可以用于定性检测 ,或作为常规PCR定性检测后的确证实验方法。     

The development and standardization of testing methods for genetically modified organisms and their derived products

As the worldwide commercialization of genetically modified organisms (GMOs) increases and consumers concern the safety of GMOs, many countries and regions are issuing labeling regulations on GMOs and their products. Analytical methods and their standardization for GM ingredients in foods and feed are essential for the implementation of labeling regulations. To date, the GMO testing methods are mainly based on the inserted DNA sequences and newly produced proteins in GMOs. This paper presents an overview of GMO testing methods as well as their standardization.     

Metabonomics study of transgenic Bacillus thuringiensis rice (T2A-1) meal in a 90-day dietary toxicity study in rats

Rice is one of the most important staple foods in the world. The Cry2A gene was inserted into the rice genome to help the plant combat insects. As the unintended effects of the genetically modified (GM) organisms are the most important barriers to the promotion of GM organisms, we have carried out a useful exploration to establish a new in vivo evaluation model for genetically modified foods by metabonomics methods. In this study, the rats were fed for 90 days with the GM and NON-GM rice diets. The changes in metabolites of the urine were detected using (1)H-NMR. The metabonomics were analyzed to see whether the GM rice can induce the metabolite changes in the rats' urine when compared with the NON-GM rice group. The multivariate analysis and ANOVA were used to determine the differences and the significance of differences respectively, and eventually we concluded that these differences did not have a biological significance. The conclusion of the metabonomics was comparable with that from the traditional method. As a non-invasive and dynamic monitoring method, metabonomics will be a new way of assessing the food safety of GM foods.     

实时荧光定量PCR技术在转基因食品检测领域中的应用

随着基因工程技术在农业生产中应用的深入,越来越多具有改良特征的转基因植物在全球范围内得到广泛种植,随之而来的转基因食品也迅猛发展,转基因产品大规模商业化引起了对安全性问题的担忧。为保证转基因产品标签制度的顺利实施,建立快速、准确、高通量的定量检测方法十分必要。我们综述了国内外转基因食品检测技术的研究进展,重点阐述了实时荧光定量PCR技术在转基因食品检测领域中的应用,并展望了通过构建质粒标准分子的方法来实现对更多转基因植物品系的定量检测。     

Exploitation of molecular profiling techniques for GM food safety assessment

Several strategies have been developed to identify unintended alterations in the composition of genetically modified (GM) food crops that may occur as a result of the genetic modification process. These include comparative chemical analysis of single compounds in GM food crops and their conventional non-GM counterparts, and profiling methods such as DNA/RNA microarray technologies, proteomics and metabolite profiling. The potential of profiling methods is obvious, but further exploration of specificity, sensitivity and validation is needed. Moreover, the successful application of profiling techniques to the safety evaluation of GM foods will require linked databases to be built that contain information on variations in profiles associated with differences in developmental stages and environmental conditions.     

5种转基因油菜转化体特异性多重PCR检测方法

【目的】全球转基因植物及其产品的数量和种类越来越多,迫切需要可同时精准高效检测多个转化载体的检测方法。【方法】针对RF1、MS8、Topas19/2、Oxy235和RF3等5个转基因油菜品系的侧翼序列及油菜内源基因cruciferin A(Cru A)序列设计多重聚合酶链式反应特异性引物,通过对转基因油菜、转基因大豆、转基因玉米、转基因水稻、转基因棉花等不同作物进行PCR扩增来测试所选择的引物特异性,优化多重PCR反应引物的浓度,用所建立的检测体系对不同混合比例的转基因油菜进行多重PCR扩增来测试所建立的检测方法的灵敏度。【结果】通过测试,仅在含有目标样品中检测出阳性结果,灵敏度达0.05%,表明所建立的6重PCR检测方法可同时精准检测RF1、MS8、Topas19/2、Oxy235和RF3等5种转基因油菜转化载体。【结论】所建立的6重转基因油菜转化体特异性PCR检测方法通量高、特异性好、灵敏度高,符合有关转基因产品检测的要求,可作为转基因油菜检测的有效方法。     

转基因食品DNA提取研究进展

为了满足消费者对转基因食品的知情权,建立准确、快速、高效的转基因成分检测技术至关重要,而高质量DNA模板的获取,是转基因食品进行基因检测的前提.对近几年来国内外转基因食品DNA提取方法:十六烷基三甲基溴化铵(hexadecyl trimethyl ammonium bromide,CTAB)法、十二烷基硫酸钠(dode...     

New EU legislation for risk assessment of GM food: no scientific justification for mandatory animal feeding trials

This commentary focuses on the potential added value of and need for (sub)‐chronic testing of whole genetically modified (GM) foods in rodents to assess their safety. Such routine testing should not be required since, due to apparent weaknesses in the approach, it does not add to current risk assessment of GM foods. Moreover, the demand for routine testing using animals is in conflict with the European Union (EU) Commission's efforts to reduce animal experimentation. Regulating agencies in the EU are invited to respect the sound scientific principles applied to the risk assessment of foods derived from GM plants and not to interfere in the risk assessment by introducing extra requirements based on pseudo‐scientific or political considerations.     

Detection of genetically modified organisms by electrochemiluminescence PCR method

With the development of biotechnology, more and more genetically modified organisms (GMOs) have entered commercial market. Because of the safety concerns, detection and characterization of GMOs have attracted much attention recently. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) combined with hybridization technique was applied to detect the GMOs in genetically modified (GM) soybeans and papayas for the first time. Whether the soybeans and the papayas contain GM components was discriminated by detecting the Cauliflower mosaic virus 35S (CaMV35S) promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM soybeans and papayas. The technique may provide a new means in GMOs detection due to its simplicity and high efficiency.     

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 (1)H 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.     

Not just about “the science”: science education and attitudes to genetically modified foods among women in Australia

Previous studies investigating attitudes to genetically modified (GM) foods suggest a correlation between negative attitudes and low levels of science education, both of which are associated with women. In a qualitative focus group study of Australian women with diverse levels of education, we found attitudes to GM foods were part of a complex process of making “good” food decisions, which included other factors such as locally produced, fresh/natural, healthy and nutritious, and convenient. Women involved in GM crop development and those with health science training differed in how they used evidence to categorize GM foods. Our findings contribute to a deeper understanding of how GM food, and the role of science and technology in food production and consumption more broadly, is understood and discussed amongst diverse “publics” and across different “sciences,” and to research related to deepening public engagement at the intersection of science and values.     

国际上主要国家和地区农业转基因产品的标识制度

转基因标识是表明产品含有转基因成分或者由转基因生物生产、加工而成的一种标识。随着全球转基因技术研发和应用的不断推进,国际上对农业转基因产品的标识管理更加关注与重视。通过阐述农业转基因产品标识制度的形成与发展过程,研究欧盟、美国、加拿大、日本、韩国等主要国家和地区的转基因产品标识管理制度,总结出成分关注标识、过程关注标识、自愿标识、强制性标识、定性标识、定量标识、全面标识、目录标识等不同标识类别的特点与利弊,并分析了国际上关于标识豁免及阴性标识等方面的政策规定,为我国的农业转基因产品标识管理工作提供了启示与参考。     

Analyzing consumers' preferences towards GM food in southern Spain

In Europe, since the 1990s, food safety crises have heightened public concerns about and distrust towards the agro-food sector, making consumers wary of genetically modified (GM) foods. The application of gene technology to agricultural production has become a highly controversial scientific, political and social issue due to the uncertainty about the potential risks and benefits from GM food consumption. In this regard, public views influence consumers' demands and hence determine the future of this technology's development. Biotechnology proponents attribute the current consumer resistance to GM foods to the lack of tangible benefits for the consumers with higher functional properties. Thus, this paper estimates consumers' preferences regarding GM foods by simulating a market for GM functional cornflakes in southern Spain using the conjoint analysis method. Additionally, market segmentation was performed using a cluster analysis and the consumers' profile differences for each segment were identified. The main results indicate that consumers prefer the GM-free food; nonetheless, the market segmentation distinguishes a potential market niche for GM foods and indicates that businesses can develop strategies to target these segments. Therefore, the marketing strategies implemented in southern Spain should be focused on the health functional claims of second generation GM food.     

Are all GMOs the same? Consumer acceptance of cisgenic rice in India

India has more than 215 million food‐insecure people, many of whom are farmers. Genetically modified (GM) crops have the potential to alleviate this problem by increasing food supplies and strengthening farmer livelihoods. For this to occur, two factors are critical: (i) a change in the regulatory status of GM crops, and (ii) consumer acceptance of GM foods. There are generally two classifications of GM crops based on how they are bred: cisgenically bred, containing only DNA sequences from sexually compatible organisms; and transgenically bred, including DNA sequences from sexually incompatible organisms. Consumers may view cisgenic foods as more natural than those produced via transgenesis, thus influencing consumer acceptance. This premise was the catalyst for our study—would Indian consumers accept cisgenically bred rice and if so, how would they value cisgenics compared to conventionally bred rice, GM‐labelled rice and ‘no fungicide’ rice? In this willingness‐to‐pay study, respondents did not view cisgenic and GM rice differently. However, participants were willing‐to‐pay a premium for any aforementioned rice with a ‘no fungicide’ attribute, which cisgenics and GM could provide. Although not significantly different (P = 0.16), 76% and 73% of respondents stated a willingness‐to‐consume GM and cisgenic foods, respectively.     

Development of a multiplex fluorescence quantitative PCR for detection of genetically modified organisms

The commercially available genetically modified plants authorized worldwide and therefore the target sequences for molecular detection of genetically modified organisms (GMOs) are ever-increasing. The European Union has implemented a set of very strict procedures for approval to grow, import and/or utilize GMOs as food or food ingredients. As a result, GMO laboratories and food production industry currently are forced to apply different methods to test raw material and complex processed food products. Three exogenous genes (the 35 s promoter of the cauliflower mosaic virus (35 s), nos terminator from Agrobacterium tumefaciens (nos), and the neomycin phosphotransferase II (nptII) gene) are commonly used in GMO detection. In this paper, a multiplex quantitative real-time PCR (qPCR) system was developed which allows simultaneously detection of the three exogenous genes in one reaction tube. The determined limits for the multiplex qPCR assays were 4 copies/reaction in maize samples. The specificity of the assays was demonstrated to be 100% according to the detection results of 23 genetically modified (GM) crops and 97 complex processed food products. The validation data show the individual PCR efficiency was accredited with negligible impacts between three detection channels in 7500 fluorescence quantitative PCR machine. These results indicate that this high-throughput multiplex qPCR method which combined with a reference gene is feasible for screening of GMOs, even for the processed food.     

Tracking genes from seed to supermarket: techniques and trends

Analytical techniques to track plant genes in the environment and the food chain are essential for environmental risk assessment, government regulation and production and trade of genetically modified (GM) crops. Here, I review laboratory techniques to track plant genes during pre-commercialization research on gene flow and post-commercialization detection, identification and quantification of GM crops from seed to supermarket. At present, DNA- and protein-based assays support both activities but the demand for fast, inexpensive, sensitive methods is increasing. Part of the demand has been generated by stringent food labeling and traceability regulations for GM crops. The increase in GM crops, changes in GM crop design, evolution of government regulations and adoption of risk-assessment frameworks will continue to drive development of analytical techniques.     

Attitudes in China about Crops and Foods Developed by Biotechnology

Transgenic Bt cotton has been planted in China since 1997 and, in 2009, biosafety certificates for the commercial production of Bt rice and phytase corn were issued by the Chinese government. The public attitude in China toward agricultural biotechnology and genetically modified (GM) crops and foods has received considerable attention worldwide. We investigated the attitudes of consumers, Bt cotton farmers and scientists in China regarding GM crops and foods and the factors influencing their attitudes. Data were collected using interview surveys of consumer households, farmer households and scientists. A discrete choice approach was used to elicit the purchase intentions of the respondents. Two separate probit models were developed to examine the effect of various factors on the choices of the respondents. Bt cotton farmers had a very positive attitude because Bt cotton provided them with significant economic benefits. Chinese consumers from developed regions had a higher acceptance and willingness to pay for GM foods than consumers in other regions. The positive attitude toward GM foods by the scientific community will help to promote biotechnology in China in the future. Our survey emphasized that educational efforts made by government officials, the media and scientists can facilitate the acceptance of GM technology in China. Further educational efforts will be critical for influencing consumer attitudes and decisions of government agencies in the future. More effective educational efforts by government agencies and public media concerning the scientific facts and safety of GM foods would enhance the acceptance of GM crops in China.     

Selective delivery of interleukine-1 receptor antagonist to inflamed joint by albumin fusion

Background

There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device.

Results

Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation.

Conclusions

LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.