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.     

Quantitative competitive PCR for the detection of genetically modified organisms in food

Present polymerase chain reaction (PCR) detection methods only allow the qualitative detection of GMO in food without quantitation of the GMO content. Clearly, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. This quantitative competitive (QC) PCR was first described in the early nineties and is widely used nowadays.

We have developed and evaluated QC–PCR systems for the quantitative detection of Roundup Ready^TM soybean (RRS) and Maximizer maize (MM) in food samples. Three DNA fragments differing from the GMO specific sequences by DNA insertions were constructed and used as internal standards in QC–PCR. These standards were calibrated by co-amplifying with mixtures containing defined amounts of RRS DNA and MM DNA, respectively. The calibrated QC–PCR systems were applied to several commercial food samples containing RRS and to three certified RRS flour mixtures (Fluka standards). Recently, quantitative methods for the detection of RRS were successfully tested in a collaborative study involving twelve European control laboratories. Thus, QC–PCR methods will allow to survey “de minimis thresholds” of GMOs in food.     

Quantitative analysis of genetically modified organisms (GMO) in processed food by PCR-based methods

Two different PCR-based approaches for the quantitative analysis of genetically modified organism (GMO) – components in foods are presented using Soybean derived samples as an example. The first method – a double competitive PCR – is well suited to determine threshold levels of GMO content in food. The other – PCR on-line measurement – is suited to determine ratios of transgenic versus non-transgenic component. Both methods provide a means to alleviate the problems of standardisation encountered with simple qualitative PCR approaches and will allow to cope with threshold levels for GMO, once issued by legislative bodies.     

Multiplex qualitative PCR assay for identification of genetically modified canola events and real-time event-specific PCR assay for quantification of the GT73 canola event

Genetically modified (GM) canola is the most widely grown oilseed crop in Canada. At this time, commercially produced GM canola cultivars in Canada have the events GT73/RT73 and Ms8xRf3. Commercial seed sale of canola cultivars containing the GM events such as OXY235 and T45 has been discontinued. Adventitious presence of GM seeds and grains in non-GM grains is a concern for international grain trade, and development of effective detection methods is important. A multiplex qualitative PCR procedure was established for the detection of the GM canola events OXY235, Ms8xRf3, T45 and GT73. The presence of the GM canola events was also successfully detected in ground spiked wheat and barley grain samples prepared at 0.1%, 0.5% and 1.0% levels (w/w). The GT73 real-time PCR assay was successfully used to quantify DNA extracted from spiked ground canola samples consisting of 5%, 1%, 0.5% and 0.1% GT73 (w/w).     

Transboundary movement of genetically modified organisms in India: Current scenario and a decision support system

Genetically modified (GM) crops have benefited global agriculture by introduction of superior traits for better agronomic performance, ensuring nutritional security and mitigating climate change. In India, to meet the demand of burgeoning population and to withstand the changing climate, GM crops would play an important role. Since 1997, GM crops are being imported through Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, the designated nodal organization for quarantine processing and import of GMOs (referred to GM planting material in present context) for research purposes. In the present study, an attempt has been made to analyze the trend of import of GMOs. Till the end of 2015, 205 consignments of fifteen GM crops have been imported from 19 countries by public and private sector. Detailed analysis of diversity in traits of imported GM events and imported stacked traits in cotton and maize has been made. In the recent past, four consignments of GMOs have been exported for research purposes. Involvement of public/private sector in transboundary movement of GMOs was evaluated. Along with quarantine processing of imported/exported GMOs, molecular testing for specific transgenic elements as claimed by the importer/exporter is also carried out employing polymerase chain reaction (PCR) and real-time PCR based markers. Efficient detection strategies based on GMO matrix as a decision support system, loop-mediated isothermal amplification and multi-target real-time PCR-based systems have been developed. The data presented herein would provide a decision support system to check for authorized/unauthorized GMOs in food and supply chain.     

DNA检测技术及其在微生物采油中的应用

专题论文。第一节简述了聚合酶链式反应(PCR)技术的特点、原理及在微生物采油中的作用。第二节介绍了菌种16S rRNA基因碱基序列分析的操作程序，包括基因扩增及精翻、16S rRNA基因克隆、荧光标识基因序列PCR扩增、基因碱基序列及微生物属种确定，给出了2个MEOR菌的基因碱基序列及属种。第三节介绍了多酶切和混合酶切两种PCR．RFLP基因检测技术(RFLP：限制性片段长度多态性)，后一种方法使操作减少l／4。第四节介绍了直接DNA扩增基因检测技术，包括平板菌落法(检测时间2d)和最大可能数法(当天得检测结果)，DNA模板制备及其反应体系，电泳法检测及菌种检出。第五节介绍了DNA基因检测技术在吉林油田多项微生物采油矿场试验中的应用，包括常在菌对微生物采油影响分析．微生物单井吞吐、微生物清防蜡、微生物调驱效果分析，多功能案采油菌构建的实验探索。图2参9。     

Development and in-house validation of a competitive ELISA for the quantitative detection of gluten in food

Wheat gluten contains peptide sequences, which activate specific T cells causing a chronic inflammation of the small intestine in celiac disease patients. It is well established that next to wheat gluten, the gluten-like proteins in barley and rye are similarly harmful to celiac disease patients whereas oat is generally considered safe. This study focuses on the development of an ELISA method for the detection of native and processed gluten proteins. The developed test utilizes a monoclonal antibody specific for the DQ2.5-glia-α3 T cell epitope present in the α-gliadins, which are part of wheat gluten that triggers celiac disease. The developed competitive ELISA uses a synthetic DQ2.5-glia-α3 peptide standard for calibration. The conversion from the measured DQ2.5-glia-α3 peptide concentration to gliadin content is achieved by using the experimentally determined multiplication factor of 250. The gluten content can be then calculated by multiplying the gliadin concentration by a factor of 2. A simple sample preparation method with 60% ethanol is used to extract the disease-causing proteins from cereals and processed foods. The assay was found to be specific for the detection of gluten from wheat, barley and rye with no cross-reaction with 8 tested oat varieties. The LOD and LOQ for gliadin were calculated based on the results obtained for 60 blank oats samples and they were 2.9 and 3.6 ppm, respectively. The assay could detect as little as 0.01% wheat gluten and gluten-like proteins from rye and barley in oats. The ELISA was also found to be applicable to the analysis of a range of processed food such as sauces, beers, soups and bread. In conclusion, the developed assay is a sensitive, specific and cost-effective tool for screening cereals and processed foods for the presence of harmful wheat gluten and gluten-like proteins from barley and rye.