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.     

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.     

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.     

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.     

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.     

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.     

Clarifying the regulation of genome editing in Australia: situation for genetically modified organisms

Australia’s gene technology regulatory scheme (GT Scheme) regulates activities with genetically modified organisms (GMOs, organisms modified by gene technology), including environmental releases. The scope of regulation, i.e. what organisms are and are not regulated, is set by the Gene Technology Act 2000 (GT Act) and GT Regulations 2001 (GT Regulations). The GT Act gives broad, overarching definitions of ‘gene technology’ and ‘GMO’ but also provides for exclusions and inclusions in the GT Regulations. Whether organisms developed with genome editing techniques are, or should be, regulated under countries’ national GMO laws is the subject of debate globally. These issues are also under active consideration in Australia. A technical review of the GT Regulations was initiated in 2016 to clarify the regulatory status of genome editing. Proposed draft amendments are structured around whether the process involves introduction of a nucleic acid template. If agreed, amendments would exclude from regulation organisms produced using site directed nuclease (SDN) 1 techniques while organisms produced using oligonucleotide mutagenesis, SDN-2 or SDN-3 would continue to be regulated as GMOs. The review of the GT Regulations is still ongoing and no legislative changes have been made to the GT Regulations. A broader policy review of the GT Scheme was undertaken in 2017–2018 and as a result further work will be undertaken on the scope and definitions of the GT Act in light of ongoing developments.

     

Cultivation of genetically modified organisms: resource needs for monitoring adverse effects on biodiversity

Genetically modified organisms (GMO) in non-European countries are introduced into the agro-environment on large scale with little knowledge of adverse effects on biodiversity. In the European Union (EU) possible effects of GMOs on biodiversity have to be accurately and precisely monitored. Monitoring biodiversity with a high precision is expensive and may only be achieved in close cooperation between GMO monitoring and general biodiversity monitoring. The EuMon project sampled metadata on biodiversity monitoring in Europe. Basing on the metadata, we estimated resource needs for biodiversity monitoring as needed for detecting potential adverse effects of GMOs on biodiversity. On average the analyzed schemes with a potential to detect at least a 5% change of biodiversity monitor 242.6 ± 105.4 sites at 322.6 ± 172.1 person days employing 63 ± 23 persons per year. The time invested in monitoring, given as person days, however, differed greatly between schemes and species groups, so that real manpower might be considerably higher.     

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.     

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.     

Testing for genetically modified organisms (GMOs): Past, present and future perspectives

This paper presents an overview of GMO testing methodologies and how these have evolved and may evolve in the next decade. Challenges and limitations for the application of the test methods as well as to the interpretation of results produced with the methods are highlighted and discussed, bearing in mind the various interests and competences of the involved stakeholders. To better understand the suitability and limitations of detection methodologies the evolution of transformation processes for creation of GMOs is briefly reviewed.     

Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray

In this research, we developed a multiplex polymerase chain reaction (multiplex-PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a consecutive reaction to detect a genetically modified organism (GMO). There are a total of 20 probes for detecting a GMO in a DNA microarray which can be classified into three categories according to their purpose: the first for screening GMO from un-transgenic plants based on the common elements such as promoter, reporter and terminator genes; the second for specific gene confirmation based on the target gene sequences such as herbicide-resistance or insect-resistance genes; the third for species-specific genes which the sequences are unique for different plant species. To ensure the reliability of this method, different kinds of positive and negative controls were used in DNA microarray. Commercial GM soybean, maize, rapeseed and cotton were identified by means of this method and further confirmed by PCR analysis and sequencing. The results indicate that this method discriminates between the GMOs very quickly and in a cost-saving and more time efficient way. It can detect more than 95% of currently commercial GMO plants and the limits of detection are 0.5% for soybean and 1% for maize. This method is proved to be a new method for routine analysis of GMOs.     

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。     

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。     

Methodologies and special considerations for environmental risk analysis of genetically modified aquatic biocontrol organisms

Genetic biocontrol of invasive aquatic species proposes to introduce, for control purposes, a genetically modified (GM) version of an invasive fish species to a targeted aquatic environment. Safe deployment and long term use of such technologies will depend on identifying and managing possible unintended effects to the natural environment. Environmental risk analysis (ERA) is a method for identifying the likelihood and consequences of unintended impacts, and for developing risk management strategies. For the unique situation of genetically modified biocontrol organisms (GMBOs), we review the latest thinking in ERA methodologies for GM fish and explore how terminology and assumptions from ERAs of traditional, non-modified biocontrol organisms and GM fish will need to be recast in ERAs of GMBOs. We also outline some special considerations that an ERA of a GMBOs will have to contend with: non-intuitive potential hazards; uncertainty introduced by extrapolating from domestic systems to natural ecosystems; redundancy in risk management options; and challenges of stakeholder engagement related to new technologies.