转基因棉花MON88913转化体特异性定性、定量PCR检测方法

本文以我国批准商业化的转基因耐草甘膦棉花MON88913为研究对象,建立并验证了其转化体特异性定性、定量PCR检测方法.建立的定性PCR方法的检测极限是20个拷贝棉花单倍体基因组DNA,定量PCR方法的检测和定量极限分别是10和20个拷贝棉花单倍体基因组DNA.同时,我们组织了实验室5位研究人员对建立的定量PCR检测方法进行了协同验证.对5个盲样的定量分析结果显示与真实值的偏差介于1.59% 和10.12%之间,完全满足国际标准25%偏差范围的要求,完全可用于转基因棉花MON88913的实际样品检测.     

Development of a rapid detection method for genetically modified rice using the ultra-fast PCR system

Food Science and Biotechnology - Genetically modified (GM) rice varieties containing traits such as tolerance to abiotic stress and resistance against pests and diseases continue to be developed....     

转基因大米及其安全性评价研究进展

近年来,转基因稻米的迅速发展引起了广泛关注,尤其是与食品密切相关的营养与安全问题。分析转基因大米的基因来源及对大米品质的影响,总结转基因大米可能存在的危害及其安全性评价的方法,可使人们科学的对待转基因大米,并为研究与开发新型转基因大米提供参考。     

Duplex polymerase chain reaction method for detection of unapproved genetically modified tomato (Solanum lycopersicon L.) with cucumber mosaic virus (CMV) satellite RNA gene

The complete sequence of the cucumber mosaic virus (CMV) satellite RNA (satRNA) gene that was controlled by the cauliflower mosaic virus (CaMV) 35S promoter (P-35S) and the Agrobacterium nopaline synthase terminator (T-nos) was first identified in an unapproved genetically modified (GM) tomato (Solanum lycopersicum L.), and a duplex polymerase chain reaction (PCR) method was developed based on the CMV satRNA nucleotide sequence. To detect the unapproved GM tomato, the metallocarboxypeptidase inhibitor (Mcpi) gene was selected as an endogenous reference gene for tomato and was validated using 13 different crops. The primer pair PTD-F/R was designed to amplify the junction sequences between the 35S promoter and CMV satRNA gene introduced in the unapproved GM tomato, and its specificity was also validated using several different GM events. The detection limit of the duplex PCR method is approximately 1 copy. Using the duplex PCR method, 35 processed tomato foods and 13 tomato seeds were analyzed. Of these samples, 2 GM tomato seeds were identified using the duplex PCR method. The results indicate that this duplex PCR method could be useful for detecting the unapproved GM tomato.     

Event-specific qualitative and quantitative PCR detection of genetically modified rapeseed Topas 19/2

The herbicide-tolerant transgenic rapeseed Topas 19/2 (synonym HCN92) has been approved for environmental release in Canada, Japan, Australia and the USA, and exported to a number of other countries as raw material. The purpose of this study was to establish event-specific qualitative and quantitative detection methods for Topas 19/2. The 3′-integration junction sequence spanning the host plant DNA and the integrated transgene of the Topas 19/2 event was isolated and identified. The event-specific qualitative detection method was established to produce an amplicon of 110 basepairs (bp) with an absolute detection limit of 10 initial template copies. The event-specific quantitative detection method was developed with the limit of detection (LOD) and limit of quantification (LOQ) being approximately 5 and 50 initial template copies, respectively. The developed real-time PCR systems were assessed using two mixed rapeseed samples with known Topas 19/2 contents. Expected results were obtained.     

转基因食品的检测方法及其应用与发展

随着转基因食品的迅速发展和普及,与其安全性相关的检测技术也日益受到人们的重视。针对目前国内外转基因食品的检测技术进行系统分类,简要介绍了各技术的原理、优缺点及应用情况,同时,对其面临的问题及未来发展方向做了初步探讨和展望。     

Establishment and evaluation of event-specific qualitative and quantitative PCR method for genetically modified soybean DP-356043-5

With the increasing development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. The polymerase chain reaction (PCR) technique has been the mainstay for GMO detection, especially for event-specific qualitative and quantitative PCR detection methods, which have become the internationally agreed state-of-art. This paper describes the character and event-specific quantitative detection method of DP-356043-5 (356043) soybean. In this research, the flanking regions were characterized by inverse PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right and left flanking sequences. In the qualitative PCR assay, PCR systems were established with the species-specific and event-specific primers, respectively. And event-specific primers were established on both right and left flanking sequences; the limit of detection (LOD) was both 0.05% (approximates to 42 haploid genome copies). In the quantitative TaqMan real-time PCR assay, we obtained standard curves with good linearity and relatively high efficiency of PCR. All the results indicated that the established event-specific qualitative and quantitative PCR systems for 356043 soybean in this study were reliable and suitable for 356043 soybean detection in mixed samples. Besides, based on the flanking sequence information we obtained, not only the qualitative and quantitative PCR system for detecting 356043 soybean can be established, but also some other novel event-specific detection methods using gene microarray, biosensor, etc., with target sequence on them can also be developed, which have a good value for detecting 356043 soybean.     

A detection method for recombinant DNA from genetically modified potato (NewLeaf Y potato)

A detection method using the polymerase chain reaction (PCR) was developed to detect genetically modified (GM) potato (NewLeaf Y potato; NL-Y), of which the mandatory assessment has not yet been completed in Japan. The potato sucrose synthase gene was used as an internal control. We designed a primer pair to specifically detect NL-Y without false-positive results in processed potato foods infected with the potato virus Y (PVY). The DNA introduced into NL-Y using the primer pair could be detected from potato powder samples containing 0.05% NL-Y. In addition, we designed primer pairs for recognizing the CryIIIA gene to detect the NewLeaf potato (NL), NewLeaf Plus potato (NL-P) and NL-Y and for recognizing p-FMV in order to detect NL-P and NL-Y. The proposed method was applied to the detection of NL-Y in 26 processed potato foods and NL-Y was not detected in any samples.     

Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products

Numerous analytical methods, both qualitative and quantitative, have been developed to determine reliably the presence and/or the amount of genetically modified organisms (GMOs) in agricultural commodities, in raw agricultural materials and in processed and refined ingredients. In addition to the "classical" methods for DNA and protein analysis, e.g. polymerase chain reaction and enzyme linked immunosorbent analysis, certain types of GMO-containing matrices can be profiled by complementary chemical analysis methods such as chromatography and near infrared spectroscopy. This review summarises the status of the most widely used GMO analysis technologies, identifies new areas of analytical investigation and discusses current needs and future challenges.     

Detection of genetically modified rice: collaborative validation study of a construct-specific real-time PCR method for detection of transgenic Bt rice

A collaborative trial study has been conducted for validation of an extraction method and a subsequent real-time PCR for detection of a transgenic Bt rice line (‘Bt63’) in rice products originating from China. A total of 17 laboratories participated in the study and each laboratory received 16 coded samples comprising of rice grain flours, rice noodle flours and plasmid DNAs. Of the accepted results all Bt63-positive rice grain samples (0.1 or 0.05% w/w) and all rice noodle samples prepared from marketed rice products were detected correctly. The result demonstrates that ‘Bt63’ rice is detectable even at low relative mass concentrations of 0.05%. The absolute LOD determined with plasmid DNA samples showed to be at least five copies of the ‘Bt63’ target sequence. The data provided in this study show that the method is fit-for-purpose to inspect Chinese rice products for the presence of EU-unauthorised rice lines carrying the ‘Bt63’ construct.     

A detection method of CryIAc protein for identifying genetically modified rice using the lateral flow strip assay

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.     

Development and application of a selective detection method for genetically modified soy and soy-derived products.

A method has been developed to distinguish between traditional soy beans and transgenic Roundup Ready soy beans, i.e. the glyphosate ('Roundup') resistant soy bean variety developed by Monsanto Company. Glyphosate resistance results from the incorporation of an Agrobacterium-derived 5-enol-pyruvyl-shikimate-3-phosphatesynthase (EPSPS) gene. The detection method developed is based on a nested Polymerase Chain Reaction (PCR) procedure. Ten femtograms of soy bean DNA can be detected, while, starting from whole soy beans, Roundup Ready DNA can be detected at a level of 1 Roundup Ready soy bean in 5000 non-GM soy beans (0.02% Roundup Ready soy bean). The method has been applied to samples of soy bean, soy-meal pellets and soy bean flour, as well as a number of processed complex products such as infant formula based on soy, tofu, tempeh, soy-based desserts, bakery products and complex meat and meat-replacing products. The results obtained are discussed with respect to practical application of the detection method developed.     

Establishment of a loop-mediated isothermal amplification (LAMP) detection method for genetically modified maize MON88017

In this study, we developed a visual and rapid assay for the detection of MON88017 maize using the LAMP method. The LAMP method was specific for MON88017 event and takes only 40 min and the LAMP assay sensitivity is about 40 copies, which is the same level as that of conventional PCR method. LAMP amplicons can directly be detected by naked-eye inspection after adding SYBR Green I. In summary, the LAMP method is visual, faster, and more sensitive and does not need special equipment compared to the traditional PCR technique, which makes it a very higher efficiency approach for field tests and fast screening of GMO crops, especially for on-site, large-scale testing purposes in the field.