Nanoparticle-based DNA biosensor for visual detection of genetically modified organisms

Although screening of raw ingredients and food products for genetically modified organisms (GMO) may be accomplished by detecting either the exogenous DNA or the novel protein, DNA is the preferred analyte because of its superior stability during food processing. The development of DNA biosensors is of increasing importance due to the growing demand for rapid and reliable methods for GMO detection. We report the first DNA biosensor in a dry-reagent dipstick configuration for visual detection and confirmation of GMO-related sequences by hybridization within minutes. The sensor is disposable and does not require special instrumentation. It detects the 35S promoter and nopaline synthase (NOS) terminator sequences that are present in the majority of transgenic plants. The target sequences are amplified by the polymerase chain reaction (PCR) and hybridized (7min) with probes bearing oligo(dA) tail. The biotinylated product is applied to the sensor followed by immersion in the appropriate buffer. Migration of the buffer rehydrates gold nanoparticles conjugated to oligo(dT), which hybridize with the oligo(dA) tails. The hybrids are captured by immobilized streptavidin at the test zone of the sensor giving a characteristic red line due to the accumulation of the nanoparticles. The excess of nanoparticle conjugates are captured at the control zone by immobilized oligo(dA) strands. Amplified 35S or NOS DNA is detectable at 0.16nM. Soybean powder certified reference material with 0.1% GMO content is clearly detectable after 35 and 40 amplification cycles for 35S and NOS sequence, respectively. The sensor was also applied to real samples from various sources.     

QCM-based DNA biosensor for detection of genetically modified organisms (GMOs)

Development of a mass sensitive quartz crystal microbalance (QCM)-based DNA biosensor for the detection of the hybridization of CaMV 35S promoter sequence (P35S) was investigated for the screening of genetically modified organisms (GMOs). Attention was focused on the choice of the coating chemistry that could be used for the immobilization of probe sequences on the gold surface of the quartz crystal. Two immobilization procedures were tested and compared considering the amount of the immobilized P35S probe and the extent of the hybridization reaction with the target oligonucleotide. In wet chemistry procedure, the interaction between the thiol and gold for the immobilization of a thiolated probe was employed. Direct surface functionalization of piezoelectric quartz crystals were achieved in 13.56 MHz plasma polymerization reactor utilising ethylenediamine (EDA) precursors for the immobilization of amined probes. Results indicated that immobilization of a thiolated probe provides better immobilization characteristics and higher sensitivity for the detection of the hybridization reaction. The thiolated probe was used for the detection of P35S sequence in PCR-amplified DNAs and in real samples of pflp (ferrodoxin like protein)-gene inserted tobacco plants. Fragmentation of the genomic DNAs were achieved by digestion with restriction endonucleases and ultrasonication. The results obtained from the fragmented genomic DNAs demonstrated that it is possible to detect the target sequence directly in non-amplified genomic DNAs by using the developed QCM-based DNA biosensor system. The developed QCM-based DNA biosensor represented promising results for a real-time, label-free, direct detection of DNA samples for the screening of GMOs.     

Design of a DNA chip for detection of unknown genetically modified organisms (GMOs)

MOTIVATION: Unknown genetically modified organisms (GMOs) have not undergone a risk evaluation, and hence might pose a danger to health and environment. There are, today, no methods for detecting unknown GMOs. In this paper we propose a novel method intended as a first step in an approach for detecting unknown genetically modified (GM) material in a single plant. RESULTS: A model is designed where biological and combinatorial reduction rules are applied to a set of DNA chip probes containing all possible sequences of uniform length n, creating probes capable of detecting unknown GMOs. The model is theoretically tested for Arabidopsis thaliana Columbia, and the probabilities for detecting inserts and receiving false positives are assessed for various parameters for this organism. From a theoretical standpoint, the model looks very promising but should be tested further in the laboratory. AVAILABILITY: The model and algorithms will be available upon request to the corresponding author.     

Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences

Background  

The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions.     

The adaptive potential of genetically engineered organisms in nature

Most genetically engineered organisms are unlikely to pose any threat to the environment because they are already highly selected for survival under restricted conditions. Engineering for new traits in natural or semi-natural populations, however, may entail greater risks. Genetic novelty, i.e. mutation, is an important component of the evolutionary process; a small but significant proportion of natural mutations lead to improved fitness and increased competitiveness. The artificial insertion of a new trait may produce a similar effect, setting an organism on a new and unpredictable evolutionary track. The current challenge is to attain the capacity to identify the small proportion of genetically engineered organisms in which such events might occur.     

Random amplified polymorphic DNA analysis of genetically modified organisms

Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.     

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.     

Field experiments with genetically manipulated insect viruses: ecological issues

Evil Oxford scientists, bent on world domination, release virus containing scorpion poison gene into English countryside. Well, not quite; but readers of the British press last summer might be forgiven for thinking that a latter-day Central Anarchist Council had taken root among the dreaming spires. The press reports concerned field experiments near Oxford of a genetically manipulated insect baculovirus by the National Environmental Research Council's (NERC) Institute for Virology and Environmental Microbiology (IVEM). But while it is easy to smile at sensational headlines, at least some of the press reporting has been accurate and informed, and raises important ecological issues - as well as questions about the regulation of experiments with genetically manipulated organisms.     

Aphid responses to plants with genetically manipulated phloem nutrient levels

Abstract.  Aphids ( Myzus persicae , Macrosiphum euphorbiae and Aulacorthum solani ) are reared on potato plants with phloem sucrose concentrations reduced by up to two-fold by expressing the antisense of the sucrose-H⁺ symporter ( StSUT1 ) gene. The performance of My. persicae and A. solani on the antisense plants is comparable or superior to that on the wild-type plants, but Ma. euphorbiae increases more slowly on the antisense plants than on the wild-type and fails to feed from the antisense line with the lowest phloem sucrose concentration. Electrical monitoring by electical penetration graphs reveals that Ma. euphorbiae either do not locate the sieve elements or withdraw their stylets prematurely from the sieve elements of these plants. This difference between Ma. euphorbiae and the other aphid species may reflect interspecific variation in response both to the low phloem sucrose in transgenic plants and to pleiotropic effects of the transgene on the wider physiology of the plants. All aphid species perform well on plants with phloem sucrose concentrations that, when administered via chemically-defined diets, support little or no aphid growth. These results illustrate the need for caution in extrapolating conclusions reached for diet-reared aphids to aphids on plants, and demonstrate the importance of plant-based experiments for studies of the nutritional physiology of aphids.     

Multiplex polymerase chain reaction/membrane hybridization assay for detection of genetically modified organisms

To improve detection efficiency and result accuracy, four screening primer pairs, four identifying primer pairs, one common primer pair and corresponding probes were designed for the development of multiplex polymerase chain reaction/membrane hybridization assay (MPCR-MHA) for detection of the foreign genes insert in genetically modified organisms (GMOs). After detecting condition and parameter were optimized and determined, MPCR reactions were developed for amplifying several target genes simultaneously in one tube. Primers were labeled with biotin at the 5'-end; biotinylated MPCR products were detected by hybridization to the oligonucleotide probes immobilized on a membrane with subsequent colorimetric detection to confirm hybridization. The testing of screening primers can judge whether the sample contains GMOs, and that of identifying primers can further judge what kinds of trait genes are contained in the sample. We detected nine soybean samples, six maize samples, seven potato samples and two rice samples by the MPCR-MHA method; at the same time we also detected them with single PCR-MHA method. The results between two methods have good consistency.     

Identification of plant-derived genetically modified organisms in food and feed using a hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in food and feed products. The biochip contained 22 immobilized oligonucleotide probes that were intended for (1) detection of plant DNA, (2) determination of plant species (soybean, maize, potato, and rice), and (3) identification of transgenic elements, including sequences of 35S CaMV, 35S FMV, rice actin gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, and nptII marker genes. The limit of detection was 0.5% for genetically modified (GM) soybean and maize in the analyzed samples. The tests on food and feed products using the developed approach and real-time PCR showed full agreement in determination of transgenic DNA in the samples. The proposed assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of GM component based on the identified transgene.     

基因修饰小鼠在脂代谢紊乱与动脉粥样硬化研究中的新进展

近十余年来,载脂蛋白(ApoE)与低密度脂蛋白(LDL)受体 (LDLr)基因敲除小鼠已成为研究脂代谢和动脉粥样硬化最为常用的模型.在这两种小鼠模型基础上,通过与不同的转基因、基因敲除小鼠杂交,产生了多种脂代谢紊乱和动脉粥样硬化小鼠模型,为发现调控血浆脂蛋白以及动脉粥样硬化发生的机制,创造了有利条件.此外,新的严重高甘油三酯血症小鼠模型也制备成功,本文笔者研究组研究了其中的脂蛋白脂酶缺陷模型与代谢性疾病的关系,得到了许多有意义的结果.而利用不同转基因和去基因小鼠作为供体, 以及ApoE或LDL受体缺陷小鼠作为接受体的骨髓移植技术,则大大丰富了人们对于巨噬细胞中不同基因在动脉粥样硬化发生、发展和消退过程中作用的认识.动脉粥样硬化的易损斑块形成是近年来的一个研究热点,应用小鼠模型进行模拟也取得了一定的成功.然而,小鼠与人类在脂代谢和动脉粥样硬化中存在很大的种系差异,本文对此也予以评述.     

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.     

Radiosensitivity of coding and noncoding DNA sequences of various organisms]

Relationship between pyrimidine distribution patterns and radiosensitivity (Z) of DNA molecules of different species was derived by computer analysis of recurrence frequency of pyrimidine clusters. Blocking factors (beta) and Z for coding and non-coding DNA sequences of species from different taxonomic classes have been calculated within a new model. The radiosensitivity of coding DNA sequences practically does not vary whereas Z values were increased during evolution from simplest to higher organisms. The beta and Z values calculated for several groups of individual genes were shown to vary considerably.     

Rapid detection of genetically modified organisms on a continuous-flow polymerase chain reaction microfluidics

The ability to perform DNA amplification on a microfluidic device is very appealing. In this study, a compact continuous-flow polymerase chain reaction (PCR) microfluidics was developed for rapid analysis of genetically modified organisms (GMOs) in genetically modified soybeans. The device consists of three pieces of copper and a transparent polytetrafluoroethylene capillary tube embedded in the spiral channel fabricated on the copper. On this device, the P35S and Tnos sequences were successfully amplified within 9 min, and the limit of detection of the DNA sample was estimated to be 0.005 ng μl⁻¹. Furthermore, a duplex continuous-flow PCR was also reported for the detection of the P35S and Tnos sequences in GMOs simultaneously. This method was coupled with the intercalating dye SYBR Green I and the melting curve analysis of the amplified products. Using this method, temperature differences were identified by the specific melting temperature values of two sequences, and the limit of detection of the DNA sample was assessed to be 0.01 ng μl⁻¹. Therefore, our results demonstrated that the continuous-flow PCR assay could discriminate the GMOs in a cost-saving and less time-consuming way.