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.     

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.     

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.     

转基因食品安全性评价研究进展

自1996年全球转基因作物大规模商业化生产以来,转基因作物种植面积以年均10%左右的速度迅速增长,2013年种植面积已达1.75亿hm2。其在解决全球粮食问题、环境保护、提升粮食营养质量和品质、制药以及推动经济可持续发展方面展现了重要作用。但是,随着转基因商业化生产的深入,转基因技术的潜在风险性引起了社会以及国际上更广泛的关注。事实上,在转基因技术出现之初,科学家们就开始关注其安全性问题。相关国际组织（FAO、WHO、CAC、OECD等）经过数次研究制订了一系列与转基因食品安全性有关的评价原则、指南与措施等。随着转基因技术的发展,这些安全评价策略也在不断完善。我国目前已经基本建立了转基因食品的安全评价和管理体系。转基因食品在进入市场前要经过十分全面以及系统的安全性评价,包括营养学、毒理学、过敏性等方面,从而保障转基因食品的安全性。     

Accuracy of a real-time polymerase-chain-reaction assay for a quantitative estimation of genetically modified sources in food products

The accuracy of a real-time polymerase-chain-reaction assay for genetically modified sources in food products was determined using two official test systems (kits) of primers and samples. These kits were recommended by the Federal Center of State Sanitary and Epidemiological Surveillance (Russian Ministry of Health) and the European Commission. We used the following three models of thermocyclers: iCycler iQ (BioRad, United States), Rotor-Gene 3000 (Corbett Research, Australia), and DT-322 (DNA-Technology, Russia). Studies of samples that contained 1% genetically modified sources showed that the error of a quantitative assay for genetically modified sources in food products corresponds to 20-30% and does not depend on the kit type and the thermocycler model used.     

Accuracy of a real-time polymerase-chain-reaction assay for a quantitative estimation of genetically modified sources in food products

The accuracy of a real-time polymerase-chain-reaction assay for genetically modified sources in food products was determined using two official test systems (kits) of primers and samples. These kits were recommended by the Federal Center of State Sanitary and Epidemiological Surveillance (Russian Ministry of Health) and the European Commission. We used the following three models of thermocyclers: iCycler iQ (Bio-Rad, United States), Rotor-Gene 3000 (Corbett Research, Australia), and DT-322 (DNA-Technology, Russia). Studies of samples that contained 1% genetically modified sources showed that the error of a quantitative assay for genetically modified sources in food products corresponds to 20–30% and does not depend on the kit type and the thermocycler model used.     

Biosensing and monitoring of cell populations using the hydrogel bacterial microchip

Advanced development of the hydrogel bacterial microchip (HBMChip) technique is proposed. The microchip represents an array of hemispherical gel elements 0.3-60 nl in volume attached to hydrophobic glass surface and containing live immobilized microbial cells. Separate gel elements contain each up to 10(5) cells and retain them inside even while the cells are dividing. Porous structure of the gel provides easy access of nutrients and tested substances to the immobilized cells. Optical signals from the cells are easily measurable and allow monitoring of intracellular metabolism using vital fluorescent stains or engineered constructs encoding bioluminescent or fluorescent reporters. Two possible application modes of the HBMChip have been investigated, i.e. the observation of bacteria and biosensing. The dynamics of nucleic acids synthesis in growing E. coli cells has been analyzed using vital fluorescent stain SYTO 9. A special function has been suggested for evaluation of the cell growth parameters. Biosensing properties of the HBMChip have been illustrated by quantitative analysis of antibiotics and the detection of sodium meta-arsenite.     

Advances in the analysis of DNA sequence variations using oligonucleotide microchip technology

The analysis of DNA variation (polymorphisms and mutations) on a genome-wide scale is becoming both increasingly important and technically challenging. An integration of a growing number of molecular biological methods of DNA-sequence analysis with the high-throughput feature of oligonucleotide microarray-based technologies is one of the most promising current directions of research and development.     

Integration of multiple PCR amplifications and DNA mutation analyses by using oligonucleotide microchip

We have developed a method for parallel independent on-chip amplification and the following sequence variation analysis of multiple DNA regions directly using microchip with an array of nanoliter gel pads containing specific sets of tethered primers. The method has three key features. First, DNA to be amplified is enriched at gel pads by its hybridization with immobilized primers. Second, different sets of specific primers are immobilized within various gel pads, and primers are detached within gel pads just before polymerase chain reaction to enhance the amplification. A gel pad may contain an additional permanently immobilized dormant primer that is activated to carry out the allele-specific primer extension reaction to detect mutations. Third, multiple polymerase chain reactions are confined within nanoliter gel pads covered and separated from each other with mineral oil. The method was applied to simultaneously identify several abundant drug-resistant mutations in three genes of Mycobacterium tuberculosis.     

Regulatory science requirements of labeling of genetically modified food

This paper provides an overview of the evolution of food labeling in the USA. It briefly describes the three phases of agricultural development consisting of naturally occurring, cross-bred, and genetically engineered, edited or modified crops, otherwise known as Genetically Modified Organisms (GMO). It uses the Best Available Regulatory Science (BARS) and Metrics for Evaluation of Regulatory Science Claims (MERSC) to evaluate the scientific validity of claims applicable to GMO and the Best Available Public Information (BAPI) to evaluate the pronouncements by public media and others. Subsequently claims on health risk, ecological risk, consumer choice, and corporate greed are evaluated based on BARS/MERSC and BAPI. The paper concludes by suggesting that labeling of food containing GMO should consider the consumer’s choice, such as the food used by those who desire kosher and halal food. Furthermore, the consumer choice is already met by the exclusion of GMO in organic food.     

应用遗传改造的沙门菌介导肿瘤治疗的研究进展

肿瘤是机体在各种致瘤因子作用下,局部组织细胞异常增生所形成的赘生物。肿瘤治疗一直是临床上的一个难题,而放疗、化疗和手术等常规的肿瘤治疗方法均具有明显的局限性。早期研究发现某些厌氧菌或兼性厌氧菌具有抗肿瘤效应,例如兼性厌氧菌鼠伤寒沙门氏菌可以通过某些机制选择性定殖于肿瘤并抑制肿瘤生长,其应用于肿瘤治疗具有许多潜在的优势。过去的一二十年里,已有不少研究者通过遗传操作减弱沙门菌毒力,提高其定殖肿瘤的靶向性,或以减毒沙门菌作为载体向肿瘤靶向递呈各种治疗分子,并在许多动物试验中观察到遗传改造沙门菌的良好抗肿瘤效应。随着沙门菌抗肿瘤研究的不断深入,应用遗传改造的沙门菌有希望成为一条更有效的肿瘤治疗途径。本文将从沙门菌的抗肿瘤机制、遗传改造的沙门菌介导肿瘤治疗的研究进展和目前研究存在的问题等方面进行综述。     

Stimulation of bacterial DNA transformation by cattle saliva: implications for using genetically modified plants in animal feed

To investigate the likelihood of DNA transfer from genetically modified plants (GMP) to bacteria, a rescue plasmid system for Streptococcus gordonii was modified. It was applied to monitor the DNA transformation into oral and intestinal bacteria in cattle. Transformation and recombination frequency of S. gordonii was dependent on the length of the transformed DNA. Beside horse serum, cow saliva also rendered the cells competent for DNA uptake. Competence induction was completely abolished by the addition of liquid from maize silage. Competence was partially suppressed by the addition of rumen liquid. In order to study native bacteria, 724 colonies sensitive to the antibiotics were isolated from either silage or the saliva and rumen of cows. Using horse serum, silage liquid, cow saliva or rumen liquid for competence induction, the isolates failed to integrate linearized pMK110 DNA and restore antibiotic resistance. Only 6 of the colonies obtained from the teeth of a silage-fed cow were sensitive to the antibiotics. Two isolates were related to Staphylococcus warneri. They could be transformed with the model plasmid pMK110 after induction by horse serum. DNA transformation, however, was not stimulated by incubation with cattle saliva, silage or rumen liquid. The response to competence-stimulating factors seems to vary between different bacterial species. These results suggest that the probability of DNA uptake from silage of GMPs is very low.     

Phytoseiid predators of whiteflies feed and reproduce on non-prey food sources

Two phytoseiid species, Euseius scutalis (Athias-Henriot) and Typhlodromips swirskii (Athias-Henriot), are able to suppress whitefly populations on single plants and are candidate biological control agents for whiteflies such as Bemisia tabaci (Gennadius). These species can feed on pollen and insect-produced honeydew and these food sources are likely to be available in crops. If the utilization of these food types results in increased reproduction or survival, populations of predators can persist when whitefly prey is scarce or absent. We studied the impact of pollen and whitefly-produced honeydew on the life history of the two phytoseiids. Cattail pollen allowed for survival, development and reproduction of both predators. Whitefly-produced honeydew greatly increased survival of E. scutalis, allowed for development into adulthood and for a sustained low rate of oviposition. The survival of adult T. swirskii was high on cucumber leaf tissue, either with or without pollen or honeydew. Oviposition by adults and juvenile survival of T. swirskii was very low in presence of honeydew. Biological control of whiteflies may benefit from both pollen and honeydew because these non-prey food sources have a positive effect on the life history of the two predator species, especially E. scutalis.     

Development of a genetically modified bacteriophage for use in tracing sources of pollution

Bacteriophage are frequently used as biotracers to identify the source of water pollutants. Genetic manipulation of bacteriophage M13mp18 has been used to enhance this technique by creating a library in which each recombinant bacteriophage genome contains a unique identification sequence. Techniques that identify a recombinant bacteriophage by the presence of the identification sequence, including polymerase chain reaction, restriction site polymorphism and plaque hybridization, have been developed. Recombinant bacteriophage can be used to test a large number of suspected sources simultaneously. The identification sequence also eliminates confusion with natural bacteriophage present in water samples. The performance of the modified bacteriophage and the techniques were assessed in simulated field trials on a restricted site carried out under a consent for environmental release of a genetically modified organism. The techniques were also field tested at sites in northwest England using wild-type M13 bacteriophage.     

Assessment of the food safety issues related to genetically modified foods

International consensus has been reached on the principles regarding evaluation of the food safety of genetically modified plants. The concept of substantial equivalence has been developed as part of a safety evaluation framework, based on the idea that existing foods can serve as a basis for comparing the properties of genetically modified foods with the appropriate counterpart. Application of the concept is not a safety assessment per se, but helps to identify similarities and differences between the existing food and the new product, which are then subject to further toxicological investigation. Substantial equivalence is a starting point in the safety evaluation, rather than an endpoint of the assessment. Consensus on practical application of the principle should be further elaborated. Experiences with the safety testing of newly inserted proteins and of whole genetically modified foods are reviewed, and limitations of current test methodologies are discussed. The development and validation of new profiling methods such as DNA microarray technology, proteomics, and metabolomics for the identification and characterization of unintended effects, which may occur as a result of the genetic modification, is recommended. The assessment of the allergenicity of newly inserted proteins and of marker genes is discussed. An issue that will gain importance in the near future is that of post-marketing surveillance of the foods derived from genetically modified crops. It is concluded, among others that, that application of the principle of substantial equivalence has proven adequate, and that no alternative adequate safety assessment strategies are available.     

Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H

We found that, in the presence of chimeric oligonucleotides containing complementary deoxyribo- and 2'-O-methylnucleosides, a nonaribonucleotide, [5'-32P]pACUUACCUG, was cleaved specifically upon treatment with RNase H. When 3'm(UG)d(AATG)m(GAC)5' was used as a hybridization strand, pACUUACCUG was cleaved between C6 and C7 to yield pACUUAC. In the presence of 3'm(UGAA)d(TGGA)m(C)5', the nonaribonucleotide was hydrolyzed, mainly between U8 and C9, to give pACUUACCU. This method will have a variety of applications in the field of RNA engineering.     

Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H

To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

Current and future approaches using genetically modified mice in endocrine research

Genetically modified mouse models have been used widely to advance our knowledge in the field of endocrinology and metabolism. A number of different approaches to generate genetically modified mice are now available, which provide the power to analyze the role of individual proteins in vivo. However, there are a number of points to be considered in the use and interpretation of these models. This review discusses the advantages and disadvantages involved in the generation and use of different genetically modified mouse models in endocrine research, including conventional techniques (e.g., overexpression, knockout, and knock-in models), tissue- and/or time-specific deletion of target genes [e.g., Cre-loxP and short interfering (si)RNA transgenic approaches], and gene-trap approaches to undertake functional genomics. This review also highlights the many factors that should be considered when assessing the phenotype of these mouse models, many of which are relevant to all murine physiological studies. These approaches are a powerful means by which to dissect the function of genes and are revolutionizing our understanding of endocrine physiology and metabolism.