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.     

Detection of airborne genetically modified maize pollen by real-time PCR

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.     

Fate of genetically modified microorganisms in the corn rhizosphere

The fates of genetically modified (GM)Escherichia coli andPseudomonas putida in the corn rhizosphere were investigated. Under hydrophonic and sterile conditions, both bacteria grew well in the presence of root exudates used as a sole carbon source. The growth patterns of wild types and genetically modified strains ofE. coli andP. putida were similar under the conditions tested.The presence of rhizospheric microorganisms affected the survival pattern ofE. coli. In the presence of corn roots and rhizospheric microorganisms,E. coli numbers increased during the first 3 days but were later drastically reduced, probably as a result of competition with rhizospheric microorganisms for the carbon source. However, in the presence of rhizospheric microorganisms,P. putida survived better thanE. coli in the simulated corn rhizosphere.     

PCR detection of genetically modified soya and maize in foodstuffs

The detection of genetically modified foodstuffs is becoming both a food sales and legal necessity. This study reports a rapid DNA extraction/PCR-based method for the detection of genetically modified soya (GMS) and maize (GMM) in mixed samples of transgenic and unmodified soybeans and maize kernels, and a variety of processed samples including soya flour, soya protein isolates, extruded defatted soya, acid- and alcohol-precipitated soya concentrates, soya lecithin, maize grits, seasoned corn puffs and salted corn chips. The presence of GMS DNA was determined with two pairs of primers directed towards different GMS target sequences and GMM by one primer pair. In addition, a multiplex PCR reaction which utilises an internal positive control was developed for both genetically modified organisms (GMOs). Results indicated that the methods are sensitive and specific enough to detect GMS down to a level of 0.01% dry weight in single-product PCRs and 0.1% in multiplex PCRs and GMM down to 0.001% dry weight in single-product PCRs and 0.01% in multiplex PCR. The methods are considered to represent a viable route for the commercial detection of GMS and GMM in foodstuffs.     

Detection of genetically modified organisms in foods

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. In this article, protein- and DNA-based methods employing western blots, enzyme-linked immunosorbant assay, lateral flow strips, Southern blots, qualitative-, quantitative-, real-time- and limiting dilution-PCR methods, are discussed. Where information on modified gene sequences is not available, new approaches, such as near-infrared spectrometry, might tackle the problem of detection of non-approved genetically modified (GM) foods. The efficiency of screening, identification and confirmation strategies should be examined with respect to false-positive rates, disappearance of marker genes, increased use of specific regulator sequences and the increasing number of GM foods.     

Application of triplex PCR for identification of genetically modified organism in foods

A multiplex method for detection of genetically modified organism (GMO) in various foods has been developed based on PCR-identification of cauliflower mosaic virus (CMV) 35S-promoter. It allows avoiding false positive signals due to contamination of plant raw material with CMV.     

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。     

Event-specific qualitative and quantitative PCR methods for the detection of genetically modified rapeseed Oxy-235

Oxy-235 is an oxynil-tolerant genetically modified rapeseed approved for commercialized planting in Canada. The aim of this study was to establish event-specific qualitative and quantitative detection methods for Oxy-235. Both the 5'- and 3'-junction sequences spanning the plant DNA and the integrated gene construct of the Oxy-235 event were isolated, sequenced and analyzed. A 1298-bp deletion of the rapeseed genomic DNA that showed a high similarity to the mRNA sequence of Arabidopsis thaliana was found in the integration site of the insert DNA. Event-specific qualitative PCR methods were established, with one method producing a 105-bp product specific for the 5'-integration junction and the other method producing a 124-bp product specific for the 3'-junction. The absolute detection limits for the qualitative PCR were determined to be 100 initial template copies for the 5'-junction and ten for the 3'-junction. Quantitative methods were also developed that targeted both of the junction fragments. The limit of detection of the quantitative PCR analysis was ten initial template copies for either the 5'- or 3'-junction, while the limit of quantification was determined to be approximately 50 initial template copies. The real-time PCR systems so established were examined with two mixed rapeseed samples with known Oxy-235 contents and found to obtain the expected results.     

PCR—GeneScan法检测转基因产品

利用PCR－GeneScan技术检测转基因在豆和转基因玉米,该方法的特点是PCR扩增反应后,用Genescan扫描替代琼脂糖凝胶电泳检测PCR产物。用此方法共检测了35S启动子,NOS终止子和抗除草剂（草甘膦）和抗虫（Bt)4种转基因成分,结果显示,本方法比传统的PCR一琼脂糖凝胶电泳法灵敏度高,重现性好,结果易判断,为分析检测转基因产品提供了一个实用,灵敏的方法。     

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.     

转基因农作物检测技术及其应用与发展

常用的转基因检测方法可分为两个方向,一是以检测外源基因为目标,如多聚酶链式反应分析法(PCR),二是以检测外源蛋白为目标,如酶联免疫分析法(ELISA)。此外,近年来,随着世界各国对转基因生物安全问题的日益关注,还涌现出了一批新的检测方法,如微阵列分析法(microarray),色谱分析法(chroma-tography),表面等离子共振(surfaceplasmonresonance,SPR)生物传感器分析法以及近红外线光谱分析法(nearinfraredspectroscopy,NIR)等。将对各种转基因检测方法的原理、特点及研究现状做一个扼要介绍。     

Real-time PCR method using capturing oligo-immobilized PCR tubes to determine the specific gene for soybean and genetically modified soybean in food matrices

A new real-time PCR method using capturing oligo-immobilized PCR tubes is described. This method was used to detect specific genes for soybean and genetically modified (GM) soybean in food matrices. In a standard reaction using soybean genomic DNA and a capturing oligo for the lectin gene (Le1) immobilized on the tube, we examined the effects of such hybridization conditions as the location, length, and amount of the capturing oligo, and the incubation time and temperature. Under optimized conditions, the copy number of Le1 was determined in a concentration-dependent manner from soybean genomic DNA and soybean lysate (DNA 10-1000 ng, r=0.99; lysate 1-100%, r=0.99). The copy number of a Roundup Ready soybean (RRS) gene was also successfully detected in a concentration-dependent manner (1-100%, r=0.99) from GM soybean lysate, using PCR tubes with an immobilized capturing oligo for the transgene. Our data indicate that this is a rapid and simple method to determine specific genes for soybean and GM soybean in food matrices.     

Detection of genetically modified DNA in fresh and processed foods sold in Kuwait

Developments in genetic engineering technology have led to an increase in number of food products that contain genetically engineered crops in the global market. However, due to lack of scientific studies, the presence of genetically modified organisms (GMOs) in the Kuwaiti food market is currently ambiguous. Foods both for human and animal consumption are being imported from countries that are known to produce GM food. Therefore, an attempt has been made to screen foods sold in the Kuwaiti market to detect GMOs in the food. For this purpose, samples collected from various markets in Kuwait have been screened by SYBR green-based real time polymerase chain reaction (RT-PCR) method. Further confirmation and GMO quantification was performed by TaqMan-based RT-PCR. Results indicated that a significant number of food commodities sold in Kuwait were tested positive for the presence of GMO. Interestingly, certain processed foods were tested positive for more than one transgenic events showing complex nature of GMOs in food samples. Results of this study clearly indicate the need for well-defined legislations and regulations on the marketing of approved GM food and its labeling to protect consumer's rights.     

Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms

Background

The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan^® and SYBR^® Green real-time PCR chemistries. In our study four alternative chemistries: Lux?, Plexor?, Cycling Probe Technology and LNA^® were extensively evaluated and compared using TaqMan^® chemistry as a reference system.

Results

Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics.

Conclusion

Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA^® technology is an alternative to TaqMan^® when designing assays for quantitative analysis. Because LNA^® probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan^® probe is difficult or even impossible due to sequence characteristics. Plexor? on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.

     

Detection of glycoalkaloids using disposable biosensors based on genetically modified enzymes

In this work we present a rapid, selective, and highly sensitive detection of α-solanine and α-chaconine using cholinesterase-based sensors. The high sensitivity of the devices is brought by the use of a genetically modified acetylcholinesterase (AChE), combined with a one-step detection method based on the measurement of inhibition slope. The selectivity was obtained by using butyrylcholinesterase (BChE), an enzyme able to detect these two toxins with differential inhibition kinetics. The enzymes were immobilized via entrapment in PVA-AWP polymer directly on the working electrode surface. The analysis of the resulting inhibition slope was performed employing linear regression function included in Matlab. The high toxicity of α-chaconine compared to α-solanine due to a better affinity to the active site was proved. The inhibition of glycoalkaloids (GAs) mixture was performed over AChE enzyme wild-type AChE and BChE biosensors resulting in the detection of synergism effect. The developed method allows the detection of (GAs) at 50 ppb in potato matrix.     

Microbiological contamination in genetically modified animals and proposals for a microbiological test standard for national universities in Japan.

The Biosafety Committee of the Japanese Association of Laboratory Animal Facilities of National Universities (JALAN) investigated recent episodes of microbiological contamination in genetically modified mice (GMM), and the countermeasures taken when the contaminated GMM were introduced into animal facilities, by questionnaires addressed to 53 animal facilities belonging to JALAN and serological tests. Although almost all of the contaminated GMM were accepted with conditions such as rederivation after or before reception and housing in designated rooms, contamination with a spectrum of microorganisms was demonstrated in GMM transferred domestically and from abroad. In serological tests, Mycoplasma pulmonis, mouse parvovirus, and mouse encephalomylitis virus were detected in GMM transferred from domestic facilities and from abroad. The present results of the questionnaires and serological tests suggest that GMM are highly and widely contaminated with microorganisms compared with mice from commercial breeders. Thus, we propose a microbiological requirement, including microbiological status--excellent, common, and minimum--as a guide for the transfer and procurement of mice and rats in Japan.     

Maize plants infestation by Fusarium spp. and deoxynivalenol in genetically modified corn hybrid and traditional maize cultivars

The objective of the performed investigations was to isolate pathogenic fungi from contaminated maize cobs, to assess the appearance of maize cob fusariosis and to determine grain contamination with deoxynivalenol in the cultivation of genetically modified maize containing a gene resistance against European corn borer (Ostrinia nubilalis Hbn) as well as selected non-modified cultivars. The plant material comprised the following genetically modified maize cultivar: DKC 3421 YG (MON 810) and non-modified cultivars obtained from Smolice Plant Breeding Ltd., IHAR Group: Junak (FAO 210-220), Prosna (FAO 220), SMH (FAO 230), Baca (FAO 220). Prior to harvesting, the occurrence of maize cob fusariosis was determined in the 89 (BBCH) developmental ripening stage. Microbiological assessment was carried out on grains selected from cobs characterized by various pathological symptoms. In 2008, a total of 133 isolates was obtained from the examined samples of infected maize plants, of which 51 isolates were species-identified, while in 2009, the total of 123 isolates were determined, of which 63 were species-identified. In both experimental years, the majority of isolates contained fungi from the Fusarium genus. The performed analysis of mean levels of cob contamination by fusarioses revealed that DKC 3421 YG (MON 810) and SMH (FAO 230) cultivars showed the smallest levels of contamination as well as the lowest percent of cob contamination per plant, while Junak (FAO 210-220) and Baca (FAO 220) cultivars were characterized by the highest degree of contamination. The lowest deoxynivalenol concentrations were determined in years 2008 and 2009 in the case of the DKC 3421 YG (MON 810) cultivar, whereas Prosna (FAO 220) cultivar was characterized by the highest deoxynivalenol concentration.     

Detection of exogenous genes in genetically modified plants with multiplex polymerase chain reaction

To detect exogenous genes in genetically modified plants, we have designed a multiplex polymerase chain reaction (MPCR) system that includes 7 primer pairs. With this method, the promoter, terminator, selection gene, reporter gene, and function gene can be detected simultaneously. The concentrations of each primer were optimized according to AT/TA%. Several genes in plasmid PBI121, genetically modified tobacco, and imported soybeans were detected, and the results were consistent with standard PCR. The method was sensitive enough to detect a single copy sequence.     

Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1) and LS28×Cry1Ac1 (event L/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line, a leaf folder-resistant single trait GM line, and a rice leaf blast-resistant and leaf folder-resistant stacked GM hybrid line, respectively. The FISH signals were clearly detected on the arms of one homologous chromosome pair for LS28, and on the arms of another chromosome pair for Cry1Ac1 when using the transformation vector pSBM AtCK containing the rice leaf blast-resistant gene (LS28) and pMJ-RTB containing the leaf folder-resistant gene (mCry1Ac1) as a probe, respectively. As expected, we detected two pairs of FISH signals, each on the arms of different chromosome pairs in the stacked GM rice line LS28×Cry1Ac1 when using both pSBM AtCK and pMJ-RTB as probes. These results indicate that the transgenes are located at specific homologous loci and show position stability among generations in both single trait and stacked GM rice lines. The usefulness and the necessity of FISH to detect inserted genes in transformed plants will be discussed for the purpose of future studies to develop breeding programs and conduct risk assessment of GM plants.