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.     

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.     

Identification of genetically modified vegetable sources in food and feed using hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by the hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in feed and food products. The biochip contained 22 immobilized probes intended for (i) detection of plant DNA; (ii) plant species determination (soybean, maize, potato, rice); (iii) identification of transgenic elements, including 35S CaMV, 35S FMV, rice actine gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, nptII marker genes. The limit of detection was 0.5% of genetically modified (GM) soybean and maize in analyzed samples. Identification of transgenic DNA in food and feed products using either the developed approach or real-time PCR led to virtually identical results. The assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of the GM component based on the identified transgene.     

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.     

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.     

An oligonucleotide array to detect genetically modified events in potato

For rapid and simultaneous detection of transgenic elements in genetically modified (GM) food crops, we explored DNA array technology. Forty-four oligonucleotide 23-to 31-mers were selected to use in an array on the basis of melting temperature and sequence specificity. Selected oligonucleotides consisted of DNA fragments corresponding to structural and regulatory elements and selectable markers used in developing transgenic crops, such as potato. Other oligonucleotides represented endogenous genes from potato to serve as positive controls and from heterologous crops, such as soybean and canola, to serve as negative controls. Amino-terminated oligonucleotides were hand-spotted on activated nylon membrane with a commercial spotting device. Target DNA was isolated from foliage of transgenic and nontransgenic crops, including potato, and labeled with digoxigenin-dUTP by random priming following restriction digestion to reduce DNA fragment size. Hybridization signals were visualized by an alkaline phosphatase anti-DIG-Fab conjugate and the chemiluminescent substrate, CDP-star. We detected the presence or absence of transgenic elements in transgenic and nontransgenic potato samples. Preliminary studies demonstrated that more specific and sensitive hybridization signals were generated from an oligonucleotide probe array than from a PCR product array. We anticipate that oligonucleotide probe arrays will be useful for regulatory monitoring of transgenic events.     

Screening of 12 SNPs of CYP3A4 in a Chinese population using oligonucleotide microarray

Human cytochrome P450 3A4 (CYP34A) plays an important role in the metabolism of many endo- and xenomaterials. It also exhibits a substantial interindividual variation in enzymatic activity. It has been shown that the mutant alleles of CYP3A4 encoding inactive/decreased enzymes are largely caused by single nucleotide polymorphisms (SNPs) in the gene sequence. In the present study, with the goal of detecting the known SNPs of CYP3A4, an oligonucleotide microarray was created. A genotyping standard for this microarray was also established using constructed plasmids as standard templates. The 12 SNPs of CYP3A4 in 387 Chinese DNA samples were screened using this oligonucleotide microarray. Three heterozygous subjects of CYP3A4*/*4, 5 heterozygous subjects of CYP3A4*1/*5, 4 heterozygous subjects of CPY3A4*1/6, and 6 heterozygous subjects of CYP3A4*1/*18 were found. The genotyping results of the 18 heterozygous subjects and 12 wild-type subjects were validated by direct sequencing.     

A statistical approach to quantification of genetically modified organisms (GMO) using frequency distributions

Background

According to Regulation (EU) No 619/2011, trace amounts of non-authorised genetically modified organisms (GMO) in feed are tolerated within the EU if certain prerequisites are met. Tolerable traces must not exceed the so-called ‘minimum required performance limit’ (MRPL), which was defined according to the mentioned regulation to correspond to 0.1% mass fraction per ingredient. Therefore, not yet authorised GMO (and some GMO whose approvals have expired) have to be quantified at very low level following the qualitative detection in genomic DNA extracted from feed samples. As the results of quantitative analysis can imply severe legal and financial consequences for producers or distributors of feed, the quantification results need to be utterly reliable.

Results

We developed a statistical approach to investigate the experimental measurement variability within one 96-well PCR plate. This approach visualises the frequency distribution as zygosity-corrected relative content of genetically modified material resulting from different combinations of transgene and reference gene Cq values. One application of it is the simulation of the consequences of varying parameters on measurement results. Parameters could be for example replicate numbers or baseline and threshold settings, measurement results could be for example median (class) and relative standard deviation (RSD). All calculations can be done using the built-in functions of Excel without any need for programming. The developed Excel spreadsheets are available (see section ‘Availability of supporting data’ for details). In most cases, the combination of four PCR replicates for each of the two DNA isolations already resulted in a relative standard deviation of 15% or less.

Conclusions

The aims of the study are scientifically based suggestions for minimisation of uncertainty of measurement especially in —but not limited to— the field of GMO quantification at low concentration levels. Four PCR replicates for each of the two DNA isolations seem to be a reasonable minimum number to narrow down the possible spread of results.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0407-x) contains supplementary material, which is available to authorized users.     

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.     

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.     

Synthetic peptides as artificial receptors towards proteins from genetically modified organisms

The aim of this work was the preparation of peptide ligands with good affinity and selectivity towards proteins from genetically modified organisms, namely neomycin phosphotransferase II (Npt II) and the endotoxin Cry1A. A 12x12 combinatorial solid phase synthesis in aqueous medium was performed to prepare peptide libraries. From this library, two dipeptides with binding properties towards the chosen ligands (Pro-Lys for Npt II, K(eq) 7.59x10(4)M(-1); Trp-Gln for Cry 1A, K(eq) 4.35x10(4)M(-1)) were selected as scaffolds for the synthesis of new tetrapeptide libraries. The equilibrium constants of the newly selected tetrapeptides increased slightly respect to the dipeptides (Pro-Lys-His-Phe for Npt II, K(eq) 7.88x10(4)M(-1); Trp-Gln-Ala-Phe for Cry 1A, K(eq) 5.65x10(4)M(-1)), but selectivity towards other proteins (wheat gliadins, bovine gamma-globulins, bovine serum albumin and chicken ovalbumin) became higher. It was demonstrated that selected tetrapeptides recognised well the ligands also in presence of very complex mixtures of potentially interfering proteins, such as whole cell lysates. This approach can be considered as a general method to obtain tailor-made reagents with antibody-like binding properties towards biomacromolecules.     

遗传修饰生物体(GMOs)生态风险的监测

遗传修饰生物体(GMOs)释放的生态学风险往往要在相当长的时期内才表现出来,因而必须对其进行长期监测。监测的内容和方法依对象的不同而有所不同。在短期和长期监测中,数学模型具有重要的作用。本文就监测的内容、原则和方法进行了全面的论述。     

Technocratic precautionary principle: Korean risk governance of genetically modified organisms

Regulations for genetically modified organisms (GMOs) in Korea fluctuate between technocracy and the precautionary principle (PP). Technocratic PP denotes the coexistence, or coproduction, of technocracy with PP – a complex ensemble of technocratic, precautionary policies, and hybrids of the two. This paper analyzes four types of PP-based policies linked to Korean GMO regulations: foresight and monitoring of risk; reverse burden of proof; public participation; and the public's right to know. Korean GMO regulations are consistent with the Cartagena Protocol on Biosafety, a type of PP, but lack long-term risk assessment as well as public participation. Technocracy is embedded both in advance informed agreements as a reverse burden of proof and in proof-based GMO labeling as a right-to-know policy. Technocratic PP results in inconsistencies between PP and technocratic epistemology and the gap between PP-based institutions and technocratic practices. Technocratic PP is therefore a typical phenomenon that occurs in the “glocalization” of risk regulation.     

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。     

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20 + species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.