Detection of DNA in soybean oil

 The presence of DNA in foodstuffs which are, or contain, genetically modified organisms (GMO) is the basic requirement for labelling of GMO food in Switzerland and is also being discussed as a requirement for labelling in the European Union. The present work presents data indicating that no genetic material can be recovered after the first processing steps of soybean oil, i.e. when crude soybean oil is simply centrifuged. This fact is of some relevance because centrifugation is one of the first steps in industrial oil processing. A nested PCR system utilising a single copy gene (lectin 1) showed that centrifugation purified the oil from the genetic material by at least a factor of 10 000. The same results were observed when industrially processed oil fractions were analysed. Thus, with respect to the presence of DNA, soybean oil from GMO soybeans is identical to traditional oil and does not need to be labelled as a GMO product in Switzerland. Received: 21 January 1998 / Revised version: 30 March 1998     

Quantitative competitive PCR for the detection of genetically modified soybean and maize

 The surveillance of food labelling concerning genetically modified organisms (GMOs) requires DNA-based analytical techniques. Present assay systems allow the detection of GMO in food; however, they do not permit their quantitation. In this study, we report the development of quantitative competitive polymerase chain reaction (QC-PCR) systems for the detection and quantitation of the Roundup Ready soybean (RRS) and the Maximizer maize (MM) in food samples. Three DNA fragments that differ from the GMO-specific sequences by an insertion were constructed and used as internal standards in the PCR. These standards were calibrated by co-amplifying with mixtures containing RRS DNA and MM DNA, respectively. The calibrated QC-PCR systems were applied to nine commercial food samples containing RRS DNA and to three certified RRS flour mixtures in order to elucidate whether these food samples contain more or less than 1% RRS DNA. Finally, the GMO contents of four samples that were found to contain more than 1% RRS were determined by QC-PCR using various amounts of standard DNA. Received: 13 January 1998 / Revised version: 4 March 1998     

Development of two screening duplex PCR assays for genetically modified organism quantification using multiplex real-time PCR master mixes

Since 2001, the traceability and labelling of genetically modified organism (GMO) food and feed derived products are obligatory in the European Union. Genetically modified organisms (GMO) are commonly detected via PCR tests. These tests typically involve several steps: (1) screening (2) construct specific (3) event specific and (4) reference gene. Screening tests are based on sequences frequently used for GM development, allowing for the detection of a large number of GMOs. To improve GMO detection efficiency, using specific multiplex master mixes, we developed two real-time PCR screening duplex PCR assays for the detection of P35S/Tnos and Pnos/T35S sequences. By combining these tests, we were able to reduce the time and cost of analysis. For the Pnos/T35S duplex, good sensitivity was obtained using one of the mixes compared to the others. Both duplexes had 100% specificity when tested on DNA from GM maize, rapeseed and soybean. When the duplexes were tested on DNA containing various amounts of GM maize and soybean, the corresponding targets were detected. The detection limit of our methods was found to be between 2 and 8 haploid genome copies for both P35S/Tnos and Pnos/T35S tests. In summary, with high efficiency and good linearity, the proposed two screening duplexes allow for more efficient GMO detection.     

Monitoring genetically modified soybean along the industrial soybean oil extraction and refining processes by polymerase chain reaction techniques

In the present work, the extraction and detection of DNA along a complete industrial soybean oil processing chain was described to monitor the presence of Roundup Ready^® (RR) soybean. The analysed samples comprised all the steps prior to industrial oil extraction, namely, raw, cracked, laminated and expanded seeds, and the defatted flour as a sub-product. The samples collected at the refining unit included the crude oil, degummed/neutralised, washed, bleached and deodorised oil, as final product. The amplification of soybean lectin gene by end-point polymerase chain reaction (PCR) was successfully achieved in all the steps of extraction and refining processes, until the fully refined soybean oil. The amplification of RR soybean by PCR assays using event-specific primers was also achieved for all the extraction and refining steps, except for the intermediate steps of refining (neutralisation, washing and bleaching) possibly due to sample instability. The real-time PCR assays using specific probes confirmed all the results and proved that it is possible to detect and quantify genetically modified organisms in the fully refined soybean oil. To our knowledge, this has never been reported before and represents an important accomplishment regarding the traceability of genetically modified organisms in refined oils.     

大豆及豆制品中DNA提取方法初探

为了满足转基因食品标签制管理的需要，必须从食品中提取DNA，并运用PCR方法检测外源基因的有无。本文初探了大豆及常见豆制品中DNA的提取方法，为转基因食品的PCR检测打下了基础。     

A PCR-microarray method for the screening of genetically modified organisms

A new method to screen and to identify genetically modified organisms (GMO) is presented in this paper. It is based on the detection of multiple genetic elements common to GMO by their amplification via PCR followed by direct hybridisation of the amplicons on microarray. The pattern of the elements is then compared to a database of the composition of EU-approved GMO and an identification of the GMO is then proposed. The limit of detection of the method was ≤0.1% GMO content (w/w) expressed as the amount of target DNA present in the template for single unprocessed material. The DNA targets were detected both in reference materials and in mixtures with the same detection limit. The specificity for the detection of the different elements was found to be very good with no cross-reaction even in samples with two GMO present at different concentrations. The paper presents examples of GMO identification and discusses the potential and limitation of such approaches and how they can facilitate the work of private and enforcement detection laboratories.     

快速检测试纸条法在大豆转基因检测中的应用

中国每年进出口大量大豆产品,需要对其转基因成分进行监控,PCR检测法是目前国内外检测生物技术产品最常用的方法,但需要的仪器设备价格昂贵,检测周期较长,而快速检测试纸条法是一种快速检测方法,可以满足大豆产品在种植、加工及贸易中的监控需要,实验证明：采用快速检测试纸条测定大豆转基因含量,测定时间仅需10min,测定结果准确,与传统PCR方法相比,缩短了工作时间,提高了工作效率。     

Parallelised real-time PCR for identification of maize GMO events

Identification of specific material derived from genetically modified organisms (GMO) present in food, feed or seed samples screened positive for the presence of genetic modification(s) is mandatory for the official food and feed control in the European Union. Since the introduction of regulation (EC) No 1829/2003 in 2004, the number of maize GMO events either approved in the EU or with a pending application grew constantly. By the sheer multitude of events and crossed events (stacks), maize poses a special challenge on official food and feed control. We developed a modular qualitative detection system for the parallel identification of maize GMO events to cope with the increasing number of GMO potentially present in routine samples. This system is based on validated real-time PCR assays in a microtitre plate format grouped modularly by crop species. The maize module identifies in parallel, i.e. simultaneously, 15 maize events and RoundupReady soy in a single analytical run of approximately 2 h. Maize modules can be conveniently prepared in advance and stored at −20 °C until use. Ready-to-use reference DNA mixtures serve as positive controls. The modular approach is flexible as it allows easy change or addition of individual detection reactions, if necessary, e.g. when new validated methods become available. 23 food, 14 feed and 8 seed samples were successfully analysed with the maize module. The parallel detection of nine different GMO maize and soy events in single routine samples demonstrated the usefulness of the parallelised modular approach for routine GMO analysis.     

Quantitative detection of the 35S promoter and the NOS terminator using quantitative competitive PCR

 DNA-based analytical methods are often used to verify the presence of genetically modified organisms (GMOs) in food. In Switzerland, a preliminary study, organized by a subcommission of the Swiss Food Manual, of different polymerase chain reaction (PCR) systems for the detection of GMOs showed that the application of qualitative PCR systems can lead to interlaboratory differences of at least a factor of 10. These differences can be diminished using internal standards (competitors). The quantitative competitive (QC) PCR for the detection of the 35S promoter or the NOS terminator in food samples is presented. The GMO content of food samples can be determined using QC-PCR. Received: 2 July 1998 / Revised version: 15 October 1998     

大豆高蛋白质粉的研发

大豆高蛋白质粉是对油料蛋白进行精深加工而研制的一种新产品.主要根据目前消费市场上生产和销售的豆乳粉、豆粉、豆奶及豆浆中低档次产品情况,按照营养效价平衡配比,选用非转基因优质大豆为原料,添加了磷脂与植脂末强化剂而研发的高蛋白、高档次、高价值冲调用终端产品.工艺技术特点是对原料进行二次浸出,对凝乳和乳清进行二次分离,温度自控,连续中和,对蛋白质进行后修饰改性,使产品内在质量为蛋白质含量高,溶解性强,分散性能好.外观质量为具有牛乳一样的乳白色,口感细腻,风味宜人.研发的大豆蛋白质粉与国内外同类产品相比,质量有所提高,技术有所创新,保鲜有所进步,填补了国内空白.产品可广泛应用于保健品、食品、饮品领域,社会和经济效益明显.     

Food authentication by PCR-based methods

Food authenticity is presently a subject of great concern to food authorities, as the incorrect labelling of foodstuffs can represent a commercial fraud. The implication of misleading labelling can be much more important concerning the presence of potentially allergenic foods. The need to support food labelling has provided the development of analytical techniques for the analysis of food ingredients. In the last years, several methods based on polymerase chain reaction (PCR) have been proposed as useful means for identifying species of origin in foods, as well as food allergens and genetically modified organisms (GMO), due to their high specificity and sensitivity, as well as rapid processing time and low cost. This work intends to provide an updated and extensive overview on the PCR-based methods for food authentication, including also methods for allergens and GMO the detection in foods.     

4000t/d大豆压榨技术和设备探讨

4 000 t/d大豆压榨,预处理工艺中采取两道破碎和脱皮工艺,配备取环形刮板、新型平面回转筛、破碎机、逆流干燥器、巴西膨化机,在输送设备和溜管方面,提出选材和制造要求;在浸出方面,浸出器浸出时间42 min、料层高度1.1 m,采用8层DT、5层DC、降膜式蒸发器、立式冷凝器,增加1台油脂干燥器。调试结果表明:满负荷生产时电耗26 kW·h/t、汽耗270 kg/t;产品豆粕、四级油和浓缩磷脂质量达标。     

Monitoring of genetically modified food and feed in the Tunisian market using qualitative and quantitative real-time PCR

Genetically modified organisms (GMO) invade more and more the agricultural production in the world. Although there are no legislations on GM labeling and cultivation of GM crops in Tunisia, the present study aims to check the status of GMO in Tunisian market using qualitative and quantitative real time-PCR (QRT-PCR). Three-hundred-sixty five samples were collected and different DNA extraction methods were adapted and optimized. Specific primers targeting 35S promoter from Cauliflower mosaic virus (CaMV) and nopaline synthase terminator from Agrobacterium tumefaciens (At) were used for the detection of the GMO insert and Taxon specific primers for the detection of plant species. Validated Taqman® probes (EU-RL) targeting event specific regions of the maize events MON810, Bt11, and the soybean event RRS were used for the quantification studies. Seven food and feed products showed different amounts of RRS (1.9%), MON810 (2.1%), and Bt11 (1.6%). The results demonstrate for the first time the presence of GMO in Tunisian markets reinforcing the need for the development of accurate quantitative methods in routine analyses.     

Determination of unprocessed genetically modified soybean in foods using simplex and duplex real-time PCR with an internal standard

We developed a method using an internal standard to determine the amount of unprocessed genetically modified organisms (GMO) in foods as GMO weight per weight of total food (w/w_tf) using real-time PCR. Food samples were spiked with an internal standard, a ColE1 plasmid, and DNA was extracted from the samples using a silica membrane-type kit and analysed using real-time PCR. The relationship between the GMO amount and the copy number ratio of the transgene to ColE1 in 0.1–5% w/w_tf GM soybean powders was found to have a correlation coefficient ( r ) of 0.97. GMO was quantified in food samples spiked with GM soybean (final amount 0.5% w/w_tf GMO). The average GMO amount ranged from 0.35% to 0.63% w/w_tf. The results show that our method should be useful for quantifying unprocessed GMO in foods. We also developed a duplex assay, which is simpler and more accurate than the simplex assay.     

Applicability of the “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” in processed maize matrices

Simple tools to detect the presence of genetically modified organisms (GMO) in commercial products represent a valuable aid in managing the legal requirements for GMO testing in a cost-effective way. The “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” was developed to meet such requirements and was here further tested for its applicability on detecting GMO in recalcitrant maize matrices. Sixty-four processed maize products were purchased from the market of the European Union and analyzed for their GMO content. Seventy-five percent of the test samples were positive for the presence of GMO. In two samples, trace amounts of the so-called asynchronously authorized GMO could be detected. The overall outcome of the analyses indicated that most of the commercially available genetically modified maize events for food use could be detected in this study. Finally, the possibility to use the “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” in detecting GMO at a 0.1% mass level is documented. The implications of these results on the further development of such type of PCR-based GMO detection systems are discussed.     

Collaborative Study of a T-nos Real-Time PCR Method for Screening of Genetically Modified Organisms in Food Products

In the EU the presence of genetically modified organisms (GMO) in food is controlled by the Member States official laboratories within their national inspection and monitoring programs. The most frequently used approach to detect the presence of GMO material in different food matrices is the PCR-based screening for the CaMV 35S promoter and the nos terminator (T-nos) DNA sequence from Agrobacterium tumefaciens. A real-time PCR method for detection of T-nos and its validation in a collaborative trial study is described in this paper. The PCR system amplifies a 84 bp fragment and showed to be sensitive to detect at least 5 copies of the T-nos DNA sequence. The assay was adapted for use in real-time PCR instruments using plastic reaction vials and glass capillaries, respectively. A total of 24 laboratories participated in the study and each laboratory received 18 DNA blind samples prepared from GMO certified reference materials (0.1 % and 0.5 % NK601 maize) and from a non-GM maize flour. All samples containing NK603 maize DNA were correctly classified as T-nos positive by the participating laboratories. For the blank samples (0 % GMO) only three false-positive results were reported. A detailed evaluation of the results of the collaborative trial study is described. Received: March 19, 2007     

Collaborative Study of a T-nos Real-Time PCR Method for Screening of Genetically Modified Organisms in Food Products

In the EU the presence of genetically modified organisms (GMO) in food is controlled by the Member States official laboratories within their national inspection and monitoring programs. The most frequently used approach to detect the presence of GMO material in different food matrices is the PCR-based screening for the CaMV 35S promoter and the nos terminator (T-nos) DNA sequence from Agrobacterium tumefaciens. A real-time PCR method for detection of T-nos and its validation in a collaborative trial study is described in this paper. The PCR system amplifies a 84 bp fragment and showed to be sensitive to detect at least 5 copies of the T-nos DNA sequence. The assay was adapted for use in real-time PCR instruments using plastic reaction vials and glass capillaries, respectively. A total of 24 laboratories participated in the study and each laboratory received 18 DNA blind samples prepared from GMO certified reference materials (0.1 % and 0.5 % NK601 maize) and from a non-GM maize flour. All samples containing NK603 maize DNA were correctly classified as T-nos positive by the participating laboratories. For the blank samples (0 % GMO) only three false-positive results were reported. A detailed evaluation of the results of the collaborative trial study is described.     

PCR法检测大豆加工食品中的转基因成分

通过分子生物学手段,以聚合酶链式反应(PCR)技术为基础,建立了检测大豆加工食品中转基因成分的方法。实验分别采用CTAB法和试剂盒(Kit)法对大豆锅巴、豆浆、豆奶粉、豆腐、豆腐丝等五种大豆加工食品中的DNA进行了提取,用内标基因Lectin对此两种方法的提取效果进行了比较,并以提取的DNA为模板,利用不同的引物分别对目标基因35S和NOS进行了PCR扩增和琼脂糖凝胶电泳检测。结果表明:Kit法的DNA提取效果优于改良CTAB法,上述五种大豆加工食品中均检测出35S启动子和NOS终止子,且均含有转基因成分。     

大豆豆脐油脂品质特性的研究

为促进大豆豆脐资源的高效利用、延长大豆加工产业链,本文以大豆加工副产物——大豆豆脐为原料,采用超声辅助溶剂浸提法提取油脂,研究豆脐油脂的品质特性。结果表明：以石油醚为萃取溶剂,所得豆脐油脂酸价、过氧化值、总酚含量、角鲨烯含量、生育酚含量、植物甾醇含量分别为2.10 mgKOH/g油、0.026 g/100g、7.72 mg GAE/kg、87.63 mg/kg、4.44 mg/g、43.03 g/kg,其中,角鲨烯、生育酚和植物甾醇含量分别约为大豆油的3倍、4倍和34倍;豆脐油脂中亚麻酸含量高达22.2%,约为大豆油的2~5倍;豆脐油脂的主要甘油三酯及含量分别为LLLn(17.89%)、LLL(13.12%)、PLL(11.85%)、PLLn(11.36%)、LLnLn(9.66%)。通过对豆脐油脂品质特性分析,以期为大豆豆脐综合开发利用提供数据支撑。