Bulk acoustic wave affinity biosensor for genetically modified organisms detection

Bulk acoustic waves have been applied as affinity sensors. In particular, a nucleic acid sensor for hybridization studies has been developed and applied for detecting DNA target sequences in solution. A DNA probe is immobilized on the sensor surface while the target sequence is free in solution; the interaction between the two complementary strands (hybridization) is followed in real-time, without the use of any label. The system has been applied to analytical problems, i.e., genetically modified organisms (GMOs) detection. The probe was complementary to characteristic DNA sequences present in GMOs. The probe sequences were internal to the sequence of 35S promoter and Nos terminator that are inserted sequences in the genome of the GMO regulating the transgene expression. Two different probe immobilization procedures were characterized to improve the performances of a piezoelectric crystal DNA sensor for GMOs detection: 1) thiol-dextran-streptavidin-biotin procedure and 2) thiol-derivatized probe and blocking thiol procedure. The system has been optimized using synthetic oligonucleotides. The probe immobilization step was monitored by a surface plasmon resonance system.     

A novel detection system for the genetically modified canola (Brassica rapa) line RT73

The herbicide-tolerant genetically modified Roundup Ready canola (Brassica napus) line RT73 has been approved worldwide for use in animal feed and human food. However, RT73 Brassica rapa lines derived from interspecific crosses with RT73 B. napus have not been approved in Japan. Here, we report on a novel system using individual kernel analyses for the qualitative detection of RT73 B. rapa in canola grain samples. We developed a duplex real-time polymerase chain reaction (PCR) method to discriminate B. napus and B. rapa DNA using scatter plots of the end-point analyses; this method was able to discriminate a group comprising B. rapa and Brassica juncea from a group comprising B. napus, Brassica carinata, and Brassica oleracea. We also developed a duplex real-time PCR method for the simultaneous detection of an RT73-specific sequence and an endogenous FatA gene. Additionally, a DNA-extraction method using 96-well silica-membrane plates was developed and optimized for use with individual canola kernels. Our detection system could identify RT73 B. rapa kernels in canola grain samples enabling the accurate and reliable monitoring of RT73 B. rapa contamination in canola, thus playing a role in its governmental regulation in Japan.     

转基因食品的检测方法

随着转基因技术的快速发展,转基因作物的种类在不断增加,对转基因食品的定性、定量检测方法也在逐步完善.目前采用的转基因成分检测方法包括核酸检测和蛋白质检测,前者主要有定性PCR、定量PCR、印迹法和基因芯片技术.后者主要有ELISA和Western杂交.文章就这些检测方法进行综述,同时对其面临的问题及未来发展方向进行初步探讨和展望.     

Strategic environmental assessments for genetically modified organisms

Genetically modified crops appear to provide a promising option in finding sustainable solutions to end global hunger and poverty, but strategic decisions need to be made on how to spend limited agricultural research funds. Potentially, strategic environmental assessment (SEA) may be used as part of an environmental management system to introduce mainstreaming of environmental considerations in the policy research and priority-setting process of development organizations to help achieve international development goals. This paper sets out a possible biotechnology SEA process that integrates qualitative and quantitative assessments with a focus on risk assessment and management within the SEA and policy environmental assessment frameworks. It uses the International Association for Impact Assessment six performance criteria for SEAs: integration; sustainability; focus; accountability; participation; and iteration.     

Patents and genetically modified soybean for glyphosate resistance

The last patents protecting glyphosate herbicide tolerant soybean (Roundup Ready) expired in 2015 (US4940835, US5188642, US5804425, US5312910, US5352605, US5530196, US5627061, US5633435, US5717084, US5728925). This opens up a very big market for generic soybean. In this paper an overview of patent status will be developed which will offer records of claims, dates and owners in different world regions, including our own experience in transgenic soybean development and implementation of an open operating system will be an opportunity for applications developers from the perspective of the emerging generic transgenic crops agenda.     

转基因食品安全吗

转基因产品是否安全,始终是百姓最为关心,且迄今为止争议最多的话题之一。2009年底,我国农业部批准了两种转基因水稻、一种转基因玉米的安全证书,成为世界上首个批准主粮转基因种植的国家,转基因产品再次激起社会各界人士热议。2010年,两会     

转基因玉米MON89034、MON810、MIR162双重数字PCR定量方法的建立

针对进出口贸易中涉及较多的转基因玉米MON89034、MON810、MIR162品系,研究一套双重数字PCR(dPCR)定量检测方法,包括引物探针的序列设计和浓度,DNA模板浓度,PCR反应过程的时间、温度等。该方法的定量限为0.1%,转基因定量检测范围覆盖0.1%～100%,线性系数为0.999,精密度优于10%。该检测方法中,每个微反应体系都含有两套引物探针,分别用FAM和VIC荧光通道进行检测,能实现内外源基因的同时检测,避免同一样品因取样不一致造成的定量差异。该方法可以同时应用到市面上最常用微滴dPCR平台和3D芯片dPCR平台,且两种方法定量结果一致性好。     

Modelling testing mechanism for mitigating genetically modified wheat contamination risks

Recent discoveries of genetically modified (GM) wheat growing in farm fields across several U.S. states have renewed public worries about GM wheat contamination if GM wheat is commercialised. There would appear to be a need for research designed to identify reactive risk mitigation strategies to maintain the sustainability of grain supply chains (SCs) before any potential GM wheat contamination undermines their integrity. This research attempts to identify cost-effective testing strategies to mitigate the GM wheat contamination risks. We explicitly model the U.S. wheat supply chain in a realistic manner to embrace complexity inherent in the system. The specification of appropriate wheat handling strategies in the SC is formulated as system optimisation problems and solved using simulation. Once solved for a base scenario, sensitivity analysis is conducted on key variables that influence wheat varietal testing strategies.     

转基因产品检测技术标准存在的问题及建议

简述了目前转基因生物及产品检测技术标准中存在的问题,针对这些问题提出了合理化建议,以期对转基因生物及产品检测技术标准的制、修订有一定的参考价值.     

Hydroxylated polychlorinated biphenyl detection based on a genetically engineered bioluminescent whole-cell sensing system

The metabolites of polychlorinated biphenyls (PCBs), such as hydroxylated PCBs (OH-PCBs), have been identified as environmental contaminants. Various studies have shown that some OH-PCBs can potentially contribute to health problems. Detection of these compounds in environmental and biological samples could provide useful information about their levels and lead to a better understanding of their apparent toxicity. To that end, we have developed a whole-cell sensing system for the detection of OH-PCBs by taking advantage of the recognition of a group of related compounds, i.e., hydroxylated biphenyls, by the product of the hbpR gene in the hbp operon from Pseudomonas azelaica strain HBP1. By fusing the luxAB genes, encoding the reporter protein bacterial luciferase, to the hbp regulator-promoter sequence, a whole-cell sensing system was developed. Here, we describe the optimization and application of this whole-cell sensing system for the detection of a model compound, 2-hydroxy-3',4'-dichlorobiphenyl. A detection limit of 1 x 10(-8) M was achieved using this system. The detection of a broad range of individual OH-PCBs as well as an OH-PCB mixture was investigated. The system can detect OH-PCBs in whole serum samples in a trace amount, which is comparable to the detection of these analytes in medium alone. We envision that the method developed can potentially be employed as a rapid and sensitive way to monitor OH-PCBs for toxicological study in the laboratory, as well as a useful tool to evaluate the presence of bioavailable OH-PCBs in natural environments.     

PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.     

Autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.     

我国非转基因大豆的市场需求

如果说20年前"转基因食品"这个词对普通消费者很陌生,那么今天,转基因食品早已深入到了我们普通百姓的生活中。转基因大豆、玉米、番茄、水稻、木瓜还有一系列深加工产品,已经走上了我们的餐桌。你很难说你的生活中没有转基因食品的影子。     

非转基因食品需不需要认证

转基因食品作为一类新资源食品,又是现代生物技术的产物,当转基因食品逐渐进入人们的食物链,成为人们餐桌上的常见食品时,其食用安全性和营养质量成为消费者最关心的问题,也是食品生产者、管理者和研究者重视的问题。     

Bioluminescence immunoassay for thyroxine employing genetically engineered mutant aequorins containing unique cysteine residues

Genetically engineered one-to-one conjugates between an analyte and a protein label have been demonstrated to yield assays with better detection limits and performance characteristics than those prepared by conventional chemical conjugation methods. To date, the preparation of these conjugates has been limited to fusion techniques where a peptide analyte is fused in frame to the protein label. To further expand the range of analytes that can be detected by using genetic engineering techniques coupled with bioanalytical methods, we have employed site-directed mutagenesis to prepare one-to-one analyte-label conjugates that include nonpeptidic analytes such as drugs, vitamins, and hormones. Specifically, we have prepared mutants of the photoprotein aequorin containing single cysteine residues suitable for site-specific conjugation. Aequorin is a photoprotein that emits light at 469 nm and has been employed as a highly sensitive bioluminescent label in the development of binding assays for important biomolecules. We have performed polymerase chain reaction-based site-directed mutagenesis on apoaequorin to yield four mutant aequorins containing unique cysteine residues at positions 5, 53, 71, and 84 in the polypeptide chain for the purpose of site-specific conjugation to a model analyte. A maleimide-activated thyroxine was selected as the model analyte and site-specifically conjugated to the mutants through their unique cysteine residues. A heterogeneous assay for thyroxine was then developed by employing the genetically engineered aequorin mutants.     

Self-regulated, droplet-based sample chopper for microfluidic absorbance detection

Akin to optical beam chopping, we demonstrate that formation and routing of aqueous droplets in oil can chop a fluidic sample to permit phase sensitive detection. This hand-operated microfluidic sample chopper (μChopper) greatly reduces the detection limit of molecular absorbance in a 27 μm optical path. With direct dependence on path length, absorbance is fundamentally incompatible with microfluidics. While other microfluidic absorbance approaches use complex additions to fabrication, such as fiber coupling and increased optical paths, this self-regulated μChopper uses opposing droplet generators to passively alternate sample and reference droplets at ~10 Hz each. Each droplet's identity is automatically locked-in to its generator, allowing downstream lock-in analysis to nearly eliminate large signal drift or 1/f noise. With a lock-in time constant of 1.9 s and total interrogated volume of 59 nL (122 droplets), a detection limit of 3.0 × 10(-4) absorbance units or 500 nM bromophenol blue (BPB) (29 fmol) was achieved using only an optical microscope and a standard, single-depth (27 μm) microfluidic device. The system was further applied to nanoliter pH sensing and validated with a spectrophotometer. The μChopper represents a fluidic analog to an optical beam chopper, and the self-regulated sample/reference droplet alternation promotes ease of use.     

High-throughput double quantitative competitive polymerase chain reaction for determination of genetically modified organisms

Quantitative competitive polymerase chain reaction (PCR), especially the double competitive PCR methods (DC-PCR), have evolved as reliable approaches to quantification of genetically modified organisms (GMO) in food. However, DC-PCR is a low-throughput method because it requires titration of each sample with various amounts of a competitive internal standard, a protocol that involves several PCRs per sample followed by electrophoresis and densitometry. To address this drawback, we have developed a new method for GMO quantification, namely, a high-throughput double quantitative competitive PCR (HT-DCPCR). In HT-DCPCR, electrophoresis and densitometry are replaced by a rapid, microtiter well-based bioluminometric hybridization assay and there is no need for titration of each sample. The determination of GM soya was chosen as a model. We have constructed internal standards (DNA competitors) both for the 35S promoter sequence and for a plant-specific reference gene (lectin). The competitors have identical size and share the same primer binding sites with the target sequences but differ in a 24-bp internal segment. Each target sequence (35S and lectin) is coamplified with a constant amount (1000 copies) of the respective competitor. The four amplified fragments are hybridized with specific probes and captured on a universal solid phase to achieve simplicity and high throughput. The hybrids are determined by using streptavidin conjugated to the photoprotein aequorin. The ratio of the luminescence values obtained for the target and the competitor is linearly related to the starting amount of target DNA. The limit of quantification for the 35S promoter is 24 copies. The proposed method was evaluated by determining the GMO content of soybean powder certified reference materials. Also HT-DCPCR was compared to real-time PCR in a variety of real samples.     

Ultrasensitive reporter protein detection in genetically engineered bacteria

We demonstrate the use of laser-induced fluorescence confocal spectroscopy to measure analyte-stimulated enhanced green fluorescent protein (egfp) synthesis by genetically modified Escherichia coli bioreporter cells. Induction is measured in cell lysates and, since the spectroscopic focal volume is approximately the size of one bioreporter cell, also in individual live bacteria. This is, to our knowledge, the first ever proof-of-concept work utilizing instrumentation with single-molecule detection capability to monitor bioreporter response. Although we use arsenic inducible bioreporters here, the method is extensible to gfp/egfp bioreporters that are responsive to other substances.