No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Detection of genetically modified organism in foods
Abstract
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.
... Nowadays, there are a lot of analytical methods for determining, characterization, and verifying GMOs in crops and foods. There ae DNA-based methods like Southern blot, qualitative and quantitative PCR, and real-time PCR, and protein-based methods like Western blot, ELISA, and lateral flow strip [17,18]. Overall, GMO detection approaches based on DNA and proteins are flexible, sensitive, and accurate. ...
... Nevertheless, these methods are laborious, expensive, time-consuming and require highly qualified professionals. Conversely, spectroscopy methods are nondestructive, synchronous, and involve consistent detection instruments that are environmentally benign, quick (<1 min), low-cost, and simple to use without requiring complicated sample preparation [17]. ...
... It works on the principle of identifying the relative proportions of C-H, N-H, and O-H bonds in organic molecules. Detection of GMOs using Vis-NIR spectroscopy is not based on the detection of changes in DNA or single proteins but on identifying the changes in structural changes due to the genotype changes caused by the introduction of transgenes for target traits [17]. Previously, several research projects were successful in using Vis-NIR spectroscopy and chemometric approaches for the effective discrimination of GM crops and foods [18,[20][21][22]. ...
In recent years, the rapid development of genetically modified (GM) technology has raised concerns about the safety of GM crops and foods for human health and the ecological environment. Gene flow from GM crops to other crops, especially in the Brassicaceae family, might pose a threat to the environment due to their weediness. Hence, finding reliable, quick, and low-cost methods to detect and monitor the presence of GM crops and crop products is important. In this study, we used visible near-infrared (Vis-NIR) spectroscopy for the effective discrimination of GM and non-GM Brassica napus, B. rapa, and F1 hybrids (B. rapa X GM B. napus). Initially, Vis-NIR spectra were collected from the plants, and the spectra were preprocessed. A combination of different preprocessing methods (four methods) and various modeling approaches (eight methods) was used for effective discrimination. Among the different combinations, the Savitzky-Golay and Support Vector Machine combination was found to be an optimal model in the discrimination of GM, non-GM, and hybrid plants with the highest accuracy rate (100%). The use of a Convolutional Neural Network with Normalization resulted in 98.9%. The same higher accuracy was found in the use of Gradient Boosted Trees and Fast Large Margin approaches. Later, phenolic acid concentration among the different plants was assessed using GC-MS analysis. Partial least squares regression analysis of Vis-NIR spectra and biochemical characteristics showed significant correlations in their respective changes. The results showed that handheld Vis-NIR spectroscopy combined with chemometric analyses could be used for the effective discrimination of GM and non-GM B. napus, B. rapa, and F1 hybrids. Biochemical composition analysis can also be combined with the Vis-NIR spectra for efficient discrimination.
... The currently available methods by which to analyze GM foods can be divided into three categories. Transgene identification and quantification can be done using either the Southern blot or polymerase chain reaction (PCR) test; transgenic expression protein detection can be done using an Enzyme-linked immunosorbent assay (ELISA), Western blot, or Lateral flow strip assay (9) . The third category of enzyme activity measurement, can be done as well, although it is not widely performed. ...
... Therefore, the detection techniques for highly processed products must be improved to meet GM food labeling testing requirements. Although several techniques are applicable, PCR analysis remains the most popular and most commonly used test for GM food analysis (9) . The GM product detection primers are designed according to the sequences of regulatory and structural genes on transgenes (10) . ...
... 1. Singleplex PCR: An initial analysis was performed using singleplex PCR to test the specificity and sensitivity of designed primers. The target fragments were designed to be less than 400 bp due to the predominance of fragments of that size in processed foods (9) . The results, shown in Figure 2, indicate that designed primers yielded satisfactory results in terms of specificity and sensitivity. ...
... These interactions were later used as signal variations for sensing purposes. The second half of the sub-network is the electrochemical detection of nucleic acids and GM nucleic acids [28,[109][110][111][112]. The middle sub-network is based on the results of the electrochemical detection of nucleic acids and further enables the detection of PCR products [113][114][115][116]. Campuzano et al. [117] prepared an electrochemical sensor for DNA recognition that allows the detection of untreated clinical samples. ...
... In addition, methods that do not utilize the label [124], immobilizing the label on the electrode [125], triple magnification model technology [126], and the use of magnetic materials [127] have been tried. the electrochemical detection of nucleic acids and GM nucleic acids [28,[109][110][111][112]. The middle sub-network is based on the results of the electrochemical detection of nucleic acids and further enables the detection of PCR products [113][114][115][116]. Campuzano et al. [117] prepared an electrochemical sensor for DNA recognition that allows the detection of untreated clinical samples. ...
Since the first successful transgenic plants obtained in 1983, dozens of plants have been tested. On the one hand, genetically modified plants solve the problems of agricultural production. However, due to exogenous genes of transgenic plants, such as its seeds or pollen drift, diffusion between populations will likely lead to superweeds or affect the original traits. The detection technology of transgenic plants and their products have received considerable attention. Electrochemical sensing technology is a fast, low-cost, and portable analysis technology. This review interprets the application of electrochemical technology in the analysis and detection of transgenic products through bibliometrics. A total of 83 research articles were analyzed, spanning 2001 to 2021. We described the different stages in the development history of the subject and the contributions of countries and institutions to the topic. Although there were more annual publications in some years, there was no explosive growth in any period. The lack of breakthroughs in this technology is a significant factor in the lack of experts from other fields cross-examining the subject. Through keyword co-occurrence analysis, different research directions on this topic were discussed. The use of nanomaterials with excellent electrical conductivity allows for more sensitive detection of GM crops by electrochemical sensors. Furthermore, co-citation analysis was used to interpret the most popular reports on the topic. In the end, we predict the future development of this topic according to the analysis results.
... To confirm the successful genetic modification, researchers conduct laboratory tests using techniques like PCR, DNA sequencing, and Southern blotting. These tests validate the presence and accuracy of the inserted gene within the plant's DNA (Ahmed 2002). After laboratory testing, the next crucial step involves greenhouse testing. ...
Enhancing the resilience of plants to abiotic stresses, such as drought, salinity, heat, and cold, is crucial for ensuring global food security challenge in the context of climate change. The adverse effects of climate change, characterized by rising temperatures, shifting rainfall patterns, and increased frequency of extreme weather events, pose significant threats to agricultural systems worldwide. Genetic modification strategies offer promising approaches to develop crops with improved abiotic stress tolerance. This review article provides a comprehensive overview of various genetic modification techniques employed to enhance plant resilience. These strategies include the introduction of stress-responsive genes, transcription factors, and regulatory elements to enhance stress signaling pathways. Additionally, the manipulation of hormone signaling pathways, osmoprotectant accumulation, and antioxidant defense mechanisms is discussed. The use of genome editing tools, such as CRISPR-Cas9, for precise modification of target genes related to stress tolerance is also explored. Furthermore, the challenges and future prospects of genetic modification for abiotic stress tolerance are highlighted. Understanding and harnessing the potential of genetic modification strategies can contribute to the development of resilient crop varieties capable of withstanding adverse environmental conditions caused by climate change, thereby ensuring sustainable agricultural productivity and food security.
... While the signal is weak during the first cycles of the reaction, it rises logarithmically with the increasing number of DNA, and the signal reaches its peak at the end of the reaction. The first starting point of the fluorescent signal is related to the amount of target DNA at the beginning of the reaction Ahmed, 2002;Zhang et al., 2019). ...
The identity-determining importance of nucleic acids in living beings is a guide for reaching the desired information about food in quality control analyzes. With these goals, the popularity of Real-time PCR (Polymerase Chain Reaction) methods, which are one of the nucleic acid-based methods, is rapidly increasing due to their high reproducibility, precision and fast result production. It is thought that informative studies about the analysis using the device will provide a basis for researches on the subject. This review contains information about the studies conducted on Real-Time PCR analysis used to detect imitation / adulteration and cheating in foods. General descriptions about the operation of the Real-Time PCR methods are given. The quality control analyzes in which the method was used were classified and explanations were made about each analysis area and examples from the studies in the literature were given.
... After the approval and cultivation of various genetically modified crops in united states and Europe in recent years, nucleic acid have become an important tool in food analysis (Peano et al., 2004& Smith et al., 2005 Several identification methods were developed. These methods were classified into different groups which focusing DNA, proteins, or other specific analysis (Gachet et al., 1999, Ahmed, 2002, Elsanhoty et al., 2006. Most of official identification method, which focused on the detection of the genetically modified foods depended on DNA. ...
... F. E. Ahmed has discussed about the invention of effective and effective GMO identification technologies was prompted by law adopted across the globe to restrict the inclusion of genetically modified organisms (GMOs) in agriculture, foodstuffs, and products. Western blots, enzyme-linked immunosorbent assays, horizontal flowing sheets, Southwestern splotches, qualitative-, quantitative-, real-time-, and limiting dilution-PCR techniques, as well as other protein-and DNA-based techniques, are described in this section [27]. ...
GMOs and the usage of GM foods have resulted from the capability to regulate and change the genetic codes of alive creatures. The capacity of GM foods to improve food processing performance, upsurge customer’s loyalty, and perhaps provides fitness advantages has expedited the adoption of GM nutriments into the diets. Though, GM crops and GMOs are still a source of virtuous dispute. The utilization of genetically modified nourishments and expertise raises ethical disquiets and personal judgement, which should preferably follow the moral values defined by diet and nourishment specialists, including such benevolence, non-maleficence, fairness, and sovereign. The future of GM crops includes a variety of aspects and trends, such as increased nutritional interest in goods, strict labelling requirements, and potentially favourable economic situations in industrialised countries. This study temporarily examines the history and contextual of genetically modified foodstuffs, diving into three zones: (1) GMO labelling, (2) regulatory disquiets, and (3) industrial uses. This article investigates the relationship between specific GM food uses and ethical issues. Ethical issues were investigated in light of the “Academy of Nutrition’s and Dietetic” (AND) code of ethics, which governs the conduct of food and nutrition professionals. Overall, the numerous moral consequences of developing and eating GM goods and GMOs must be carefully considered.
... Nowadays, spectroscopy is one of the rapid, accurate, and nondestructive methods for distinguishing between GM and non-GM crops that does not require complex sample processing [11]. Spectroscopy-based GMO identification is not to detect changes in DNA or single proteins but to detect unknown structural changes due to genotype alterations generated by the introduction of transgenes for specific traits [12]. Generally, a vast number of spectroscopy methods are available for detecting structural changes in different samples, including absorption spectroscopy, photoacoustic spectroscopy, light-induced thermoelastic spectroscopy, and photothermal spectroscopy [13][14][15]. ...
The rapid advancement of genetically modified (GM) technology over the years has raised concerns about the safety of GM crops and foods for human health and the environment. Gene flow from GM crops may be a threat to the environment. Therefore, it is critical to develop reliable, rapid, and low-cost technologies for detecting and monitoring the presence of GM crops and crop products. Here, we used visible near-infrared (Vis-NIR) spectroscopy to distinguish between GM and non-GM Brassica napus, B. juncea, and F1 hybrids (B. juncea X GM B. napus). The Vis-NIR spectra were preprocessed with different preprocessing methods, namely normalization, standard normal variate, and Savitzky–Golay. Both raw and preprocessed spectra were used in combination with eight different chemometric methods for the effective discrimination of GM and non-GM plants. The standard normal variate and support vector machine combination was determined to be the most accurate model in the discrimination of GM, non-GM, and hybrid plants among the many combinations (99.4%). The use of deep learning in combination with Savitzky–Golay resulted in 99.1% classification accuracy. According to the findings, it is concluded that handheld Vis-NIR spectroscopy combined with chemometric analyses could be used to distinguish between GM and non-GM B. napus, B. juncea, and F1 hybrids.
... Today, the detection techniques available can be divided into two major categories based on their detection targets: proteins and nucleic acids. Concurrently, there are other established methods that target the physical and chemical characteristics, for example, electrochemical methods, infrared spectroscopy, biosensor technology, and mass spectrometry (Ahmed 2002;Schreiber 1999;Xiao et al. 2020). Engvall et al. (1971) reported the development of the enzyme-linked immunosorbent assay (ELISA). ...
Brassica napus L. is a vital oil crop in China. As auxiliary tools for rapeseed breeding, transgenic technologies play a considerable role in heterosis, variety improvement, and pest resistance. Research on transgenic detection technologies is of great significance for the introduction, supervision, and development of transgenic rapeseed in China. However, the transgenic detection methods currently in use are complex and time-consuming, with low output. A single nucleotide polymorphism (SNP) chip can effectively overcome such limitations. In the present study, we collected 40 transgenic elements and designed 291 probes. The probe sequences were submitted to Illumina Company, and the Infinium chip technology was used to prepare SNP chips. In the present Brassica napus transgenic detection experiment, 84 high-quality probes of 17 transgenic elements were preliminarily screened, and genotyping effect was optimised for the probe signal value. Ultimately, a transgenic detection system for B. napus was developed. The developed system has the advantages of simple operation, minimal technical errors, and stable detection outcomes. A transgenic detection sensitivity test revealed that the probe designed could accurately detect 1% of transgenic samples and had high detection sensitivity. In addition, in repeatability tests, the CaMV35S promoter coefficient of variation was approximately 3.58%. Therefore, the SNP chip had suitable repeatability in transgene detection. The SNP chip developed could be used to construct transgenic detection systems for B. napus.
Supplementary information:
The online version contains supplementary material available at 10.1007/s13205-021-03062-6.
... (Farid, 2002).PCR yöntemleri, moleküler çalışmalar içinde, modifiye ürünlerin tespitinde kullanılan en güvenilir ve hassas yöntemlerden biridir (Yuan ve diğ. 2006). ...
Öz
Transgenik bitkiler konusundaki kaygılar son yıllarda oldukça artmaktadır. Soya ve mısır
bitkileri transgenik olarak dünyada en çok ekimi yapılan bitkilerin başında gelmektedir. Bu
konudaki endişelerle ilgili olarak başta etiketleme prensipleri olmak üzere pek çok düzenleme olmakla birlikte gıda ve yem ürünlerinde genetiği değiştirilmiş organizma (GDO) içeriğini belirlemek için kullanılan en sık kullanılan yöntem, polimeraz zincir reaksiyonudur (PCR). Birçok işlenmiş gıda ve yem ürünü, çok sayıda karmaşık bitki ve hayvan kaynağından genomik DNA içerir. Bu koşullar altında, PCR ile belirli bir miktarda DNA eklendiğinde, moleküllerin sadece küçük bir kısmı amplifikasyon için uygun uzunluktaki ilgili hedefi içerebilmektedir. Bu bakımdan PCR'nin güvenilirliğini artırmak için taksona özgü bir referans (endojen kontrol) geni ile normalleştirme önemli bir basamaktır. Bu çalışmada yerel marketlerden temin edilmiş olan işlenmiş gıda ürünleri kullanılmıştır. Elde edilen DNA örneklerinden GDO PCR amplifikasyonu amacıyla uygun uzunluktaki ilgili hedef organizmaya ait gen bölgesinin kontrolü için soya ve mısır bitkilerine ait primerlerle PCR taramaları gerçekleştirilmiş ve bisküvi, kek, kraker, cips gibi işlenmiş gıda örneklerindeki PCR ürünleri bakımından eser miktardaki içeriklerin mısır unu ve soya unu örnekleri ile birlikte değerlendirilmesi yapılmıştır. Elde edilen verilerin işlenmiş gıdalardaki transgenik içeriğin belirlenmesi adına yararlı olması hedeflenmektedir.
Anahtar Kelimeler: PCR, gıda ürünleri, transgenik bitkiler, DNA
Abstract
Concerns about transgenic plants have been increasing in recent years. Soy and corn plants are the most transgenically cultivated plants in the world. Although there are many regulations, especially labelling principles, the most commonly used method used to determine the content of genetically modified organisms (GMOs) in food and feed products is the polymerase chain reaction (PCR). Many processed food and feed products contain genomic DNA from a large number of complex plant and animal sources. Under these conditions, when a certain amount of DNA is added by PCR, only a small fraction of the molecules can contain the target of the appropriate length for amplification. In this respect, normalization with a taxon-specific reference (endogenous control) gene is a crucial step to increase PCR reliability. Processed food products obtained from local markets were used in this study. PCR scans were performed with
primers of soy and maize plants for the control of the gene region of the relevant target organism of the appropriate length for GMO PCR amplification from the DNA samples obtained, and the trace amounts of PCR products in processed food samples such as biscuits, cakes, crackers, chips, cornmeal and soy flour. It was evaluated together with the samples. It is aimed that the data obtained will help determine the transgenic content in processed foods.
Keywords: PCR, food samples, transgenic plants, DNA
... The percentage of products available in the market that contains at least one GM product is high. Approximately 60 -70% of processed foods in the United States contain GM components (Ahmed 2002;Dahl, 2012;Schneider et al. 2017). More so, the use of GM plants has increased. ...
Genetically modified (GM) crops are cultivated in over 30 countries with their products and by-products imported by over 60 countries. This chapter seeks to highlight general concerns and potential lifelong effects of consuming GM plant-based food. The consumption of GM plant-based food is as risky as consuming conventional plant-based food. However, the alien genes in these products may be unstable leading to antinutritional and unintended short-term consequences. Due to the paucity of research, no long-term effects have been attributed to the lifelong consumption of these products. Nonetheless, possible lifelong health and socioeconomic effects may result from outcrossing of genes, increasing antibiotic resistance, development of new diseases, as well as potential effects on the environment and biodiversity. Biotechnology companies need to invest more in interdisciplinary research addressing the potential lifelong effects of these products. Although GM foods are safe for consumption, clarification of current risks and lifelong effects are required.
... Positive and negative control and adequate reference material provide a base for confirmation and validation of analytical procedures and for assessing and quantifying perform laboratories and methods. 15 Moreover, reference genes also are known as house-keeping genes, a species-specific target used to determine the relative quantities reflect the ratio of GMO derived targets to a reference target. 16 Zein is a constitutive gene (housekeeping genes) and is expressed under many environmental conditions and required for several basic cellular functions. ...
This study aimed to detect genetically modified maize (GMM) in seeds of eleven imported maize hybrids grown in Jordan. We used promoter 35 S and T-nos terminator for general screening of transgenic materials. Conventional PCR detected the specific events for the screening of Bt 11, MON810, and Bt176 events. Seeds of eleven maize hybrids samples showed a positive response to the 35 S promoter; nine out of eleven showed a positive response for T-nos terminator. Bt11 event was the most used in GMM seeds, where seven out of eleven samples showed positive results. Two out of eleven hybrids showed the presence of the Bt176 event; however, MON810 not detected in any of the tested hybrids. We studied the Bt11 event in imported GMM seeds in Jordan for the first time, reinforcing the need for a mandatory labeling system and a valid simple qualitative method in routine analysis of GMCs.
... GDO'lu ürünleri doğal ürünlerden ayırmak için kullanılan metodlar moleküler biyoloji teknikleri [Polimeraz zincir Reaksiyonu(PCR)] ve GDO'ya özgü bir proteinin taranması esasına dayalı metodlar [Lateral Flow Strip, Western Blot ve Enzyme-Linked Immuno Sorbent Assay (ELISA)] olmak üzere iki ana başlık altında değerlendirilebilir. 5 Protein temelli yönteminin dezavantajı sadece bir proteinin tespitine olanak sağlaması ve bundan dolayı etkin bir tarama için çok sayıda analize ihtiyaç duyulmasıdır. Moleküler teknikler ise daha geniş bir tarama alanına sahip olduklarından daha kesin bir sonuça ulaştırırlar. ...
Avrupa Birliği raporlarına göre GDO’lu yemlerle beslenen hayvanlardan elde edilen çeşitli gıdalarda GDO’lu ürünlerin kalıntılarına rastlanmamıştır. Elde edilen hayvansal gıdaların da insan sağlığına zararının olmadığı Dünya Sağlık Örgütü(WHO), Gıda ve Tarım Örgütü (FAO) ve Avrupa Gıda Güvenliği Kurumu (EFSA) tarafından desteklenmiştir. Bununla birlikte günümüzde gelişmiş ve gelişmekte olan ülkelerin birçoğu akradite edilmiş resmi metotlarla GDO’lu yemlerin tespitini yapacak yasal düzenlemeler ve değişikliklerini yapmakta ve laboratuvar alt yapılarını kurmaktadır. Bunun nedeni tüketicilerin GDO’lu yemi tüketen hayvanlardan alınan gıdaların GDO içerme kuşkusudur. Türkiye’de GDO’lu yemlerin ve ürünlerinin kullanımı ve düzenlenmesine dair ilk yasal düzenleme 2010 yılında yapılmıştır. Daha sonra Türkiye’de GDO analizleri Gıda, Tarım ve Hayvancılık Bakanlığı(GTHB) tarafından 2017 yılında yayınlanan “Yemlerin Resmi Kontrolü için Numune Alma ve Analiz Metotlarına Dair Yönetmelik” esaslarına dayanarak TÜBİTAK ve 9 Gıda Kontrol Laboratuvarı ile GTHB’ce izin verilen 23 özel gıda kontrol laboratuarı tarafından yapılmaktadır. GDO analizi yapan 32 GTHB laboratuardan üç tanesinde kantitatif olarak GDO’lu ürün analizleri yapılabilmektedir. Yemerde GDO’lu ürün analizleri DNA ekstraksiyonunu takiben GDO’lu ürün tarama (p35S, tNOS,pFMV) Real Time PCR metodu kullanılarak ISO 21569:2005 ve ISO/24276: 2005 standartlarına göre; tip belirleme ISO/24276: 2005 ve ISO/ 21569:2005 ve miktar analizleri ISO/24276: 2005 ve ISO/21570:2005 standartlarına göre Real time PCR metotları ile yapılmaktadır. Bu standartlar Türk Standartlar Enstitüsü tarafından da kabul edilip onaylanmıştır.
... The percentage of products available in the market that contains at least one GM product is high. Approximately 60 -70% of processed foods in the United States contain GM components (Ahmed 2002;Dahl, 2012;Schneider et al. 2017). More so, the use of GM plants has increased. ...
Genetically modified (GM) crops are cultivated in over 30 countries with their products and by-products imported by over 60 countries. This chapter seeks to highlight general concerns and potential lifelong effects of consuming GM plant-based food. The consumption of GM plant-based food is as risky as consuming conventional plant-based food. However, the alien genes in these products may be unstable leading to antinutritional and unintended short-term consequences. Due to the paucity of research, no long-term effects have been attributed to the lifelong consumption of these products. Nonetheless, possible lifelong health and socioeconomic effects may result from outcrossing of genes, increasing antibiotic resistance, development of new diseases, as well as potential effects on the environment and biodiversity. Biotechnology companies need to invest more in interdisciplinary research addressing the potential lifelong effects of these products. Although GM foods are safe for consumption, clarification of current risks and lifelong effects are required.
... ATAC-Seq and ChIP-Seq also provide information on the functional state of the genome, which proved useful in the identification of inserted expression elements (S6 Fig). These expression elements are commonly used in genetic engineering [50], and this approach can be applied to identify the undisclosed insertion of these elements for commercial, biomedical, or biodefense purposes. A limitation of a multidimensional DNA analysis approach is the increased cost and labor necessary to generate these data sets over WGS. ...
There is an established relationship between primary DNA sequence, secondary and ter-tiary chromatin structure, and transcriptional activity, suggesting that observed differences in one of these properties may reflect changes in the others. Here, we exploit these relationships to show that variations in DNA structure can be used to identify a wide range of geno-mic alterations in mammalian samples. In this proof-of-concept study we characterized and compared genome-wide histone occupancy by ChIP-Seq, DNA accessibility by ATAC-Seq, and chromosomal conformation by Hi-C for five CRISPR/Cas9-modified mammalian cell lines and their unmodified parent strains, as well as in one modified tissue sample and its parent strain. The results showed that the impact of genomic alterations on each of the levels of DNA organization varied depending on mutation type (insertion or deletion), size, and genomic location. The largest genomic alterations we identified included chromosomal rearrangements and deletions (greater than 200 Kb) in four of the modified cell lines, which can be difficult to identify by standard whole genome sequencing analysis. This multi-level DNA organizational analysis provides a sensitive approach for identifying a wide range of geno-mic and epigenomic perturbations that can be utilized for biomedical and biosecurity applications.
Background
Reliable and efficient methods for detecting genetically modified organisms (GMOs) in unprocessed and processed food will be essential for establishing an effective system for traceability all along the supply chain. It is important to understand the detection of GMOs following microwave treatment, which is a common processing method used in various food products such as flours. Therefore, this study aimed to detect the presence of Cauliflower mosaic virus (CaMV) 35S promoter (P-35S), Figwort mosaic virus (FMV) promoter (P-FMV), and T-NOS (nopaline synthase terminator) genetic elements in DNA samples from untreated and microwave-treated genetically modified (GM) cereal flour samples using the qualitative polymerase chain reaction (PCR) based screening method.
Methods and results
DNA was extracted from all samples, and the efficiency of the qualitative PCR screening technique was tested by the verification studies. We performed an inhibition study with plant-specific actin (ACT) gene to the effectiveness of confirming the DNA extraction method. Then, we made the confirming of the qualitative PCR system by method performance testing criteria. The high quality and quantity of the DNA extracts from untreated and microwave-treated flour samples indicated the applicability of qualitative PCR screening assays. The results showed that microwave radiation does not significantly impact the genetic element screening in flour materials.
Conclusion
Untreated and microwave-treated flour samples had amplifiable DNA for the simultaneous screening of three genetic elements. The qualitative screening tests conducted in this study produced dependable outcomes, thus, can be successfully used for monitoring in control laboratories.
United Nations Food and Agriculture Organization (FAO), World Health Organization (WHO) and European Commission (EC), define the genetically modified organism (GMO) as organisms (plants/animals/microorganisms) that “do not occur naturally through mating and/or natural recombination”. In other words, genetic material is directly manipulated with biotechnological applications, and it is aimed to separate the millennial characteristics of plants and animals in the genetic stock through selective breeding. Foods produced from transgenic plants and animals are also called genetically modified foods. A total of 525 transgenic traits have been commercialized in 32 crops so far. The most transgenation was carried out in corn (238), cotton (61), potato (49), Argentine canola (42), soybean (41), and clove (19).
Tarımsal üretim, doğal kaynaklardan insanların yaşamını devam ettirebilmesi için
gerekli olan beslenme, barınma ve giyim gibi temel gereksinimlerini gidermek
amacıyla tarım teknikleri ile mühendislik biliminden yararlanılarak yapılan birincil
üretimi ifade etmektedir. Elde edilen ürünlerin ulusal ve uluslararası pazarlara sunularak
ülke ekonomisine katma değer sağlaması, tarım sektörünün sosyoekonomik açıdan oldukça
önemli bir konumda olduğunu göstermektedir. Tarım sektörü; insanların temel ihtiyaçlarını
karşılaması, tarım ürünlerinin sanayi sektöründe hammadde olarak değerlendirilmesi, kırsal kesimlerde insanlara gelir kaynağı olması yönüyle son derece önemlidir.
Tarımsal üretimde temel amaç; toplumların ihtiyaçlarını kısa sürede giderecek şekilde
birim alanda en yüksek verimi elde etmektir. Hızlı nüfus artışı ve sanayileşmenin etkisi
ile birlikte enerjiye ve gıdaya olan talep de her geçen gün artmaktadır. Artan bu talepler,
tarımda üretimin, üretimde verimliliğin artırılmasını zorunlu kılmaktadır. Bu verim artışı
iki temel unsurla mümkündür. Bunlar; tarımsal üretim yapılan alanların büyütülmesi ya da
birim alandan sağlanan verimi yükseltmeye yönelik yapılan çalışmalardır. Ülkemizde tarımsal üretim yapılan alanlar belirli sınırlara ulaşmış olup, var olan tarım alanlarının büyütülme ihtimali azdır. Dolayısıyla bu durum birim alandan elde edilen verimin yükseltilmesine yönelik uygulamaların artırılması ve hızlandırılması gerekliliğini ön plana çıkarmıştır. Bu da ülkemizde tarım alanında faydalanılan teknolojilerin geliştirilerek hayvan gücü ve insan işgücü yerine makina kullanımına geçilmesinin önünü açmıştır. Mekanizasyon, hayvan ve insan işgücünün yerine geçen üretim girdisi durumundadır.
Bu bölümde; tarımsal mekanizasyonun kavram ve kapsamı, tarihçesi, önemi, faydaları
ve sorunları belirtilmiş, ülkemizde mekanizasyonun gelişimi konusunda bilgi verilmiş,
tarımda teknolojinin üretimde kullanımı, mekanizasyon uygulamaları ile ilgili karşılaşılan
problemler ve çözüm imkânlarının ortaya konulması amaçlanmıştır.
The establishment and maintenance of a laboratory intended for detection of genetically modified organisms (GMO) is a comprehensive process involving much more than just the spatial arrangement of the laboratory. First, the type of analysis to be performed (Protein or DNA-Based) needs to be determined. Once established, the design of the laboratory can be completed with careful attention for workflow, throughput, required instrumentation, air flow, sample handling, personnel, data management and assay development/validation. This chapter explains the details that are needed to be considered for starting a new analytical laboratory for GMO testing, whether on the small or large scale.
Tarım; bitkisel ve hayvansal ürünlerin üretilmesi, kullanımı, hazırlanması, pazarlanması
ve dağıtımını ele alan bitki ve hayvan yetiştirme sanatı ve bilimidir.
Tarım, dünyanın gıda, içecek, ilaç, kozmetik, yakacak, barınak, kumaş ve boya gibi
ihtiyaçlarını karşılar. Ekosistemin üreticileri olan bitkiler, güneş ışığından aldığı enerjiyi
diğer canlılara fotosentezle aktararak yaşamın sürdürülmesinin temel kaynağını oluştururlar.
Süslü şekilleri, muhteşem kokuları ile yüce yaratıcının sanatını sergilerler. Toprağı
bir yorgan gibi örterek, toprağın korunmasında, canlılığının sürdürülmesinde ve temiz su
hasadında önemli işlevler üstlenirler. İnsanların temel besin ihtiyaçlarının karşılanması ve
yeterli üretim son yıllarda önemli gündemler arasındadır. Kendine yeterlilik, gıda arzı, besin
hijyeni, dengeli beslenme gibi kavramlar gündemi meşgul etmektedir. Üretimi artırmak,
yeterli gıda arzı toplumsal barışı kuvvetlendirmektedir. Tarım arazilerinin korunması,
köylerin şehir standartlarına kavuşturulması, üretim planı ve desenlerinin oluşturulması,
birim alan verimliliğini artırma, katma değeri yüksek olan ürünlerde borsa oluşturma, alım
garantisi, yeni teknolojiler kullanma, pazarlama olanaklarının artırılması, kooperatifleşme
ve üretim birliklerinin oluşturulması, toplum nezdinde köylü vatandaşlarımızın itibarlarını
artırarak sosyal statü farklarının azaltılması gereklidir.
Dünyada, tarımsal üretimde ve ürün çeşitliliğinde önemli bir konumda bulunan ülkemizde;
üretimden zevk alan ve müteşebbis ruha sahip bireyleri tarımsal üretime katabilirsek
sıralamamızı daha yukarılara çekebiliriz. Bu bağlamda tarımı farklı yönlerle ele alan ve
çözüm önerileri sunan bu kitabın politika yapıcılar ve konuyla ilgili kurum, kuruluş ve
araştırmacılar için faydalı olmasını diliyoruz. İklim değişikliği, kuraklık, organik tarım,
alternatif ürünler, topraksız tarım, seracılık, sebze ve meyve üretimi, tahıllar, baklagiller,
yağlı tohumlu bitkiler, erozyon, mekanizasyon, göç, arı, ipekböceği, mantar, tıbbi aromatik
bitkiler, moleküler ve bitki doku kültürü, genetiği değiştirilmiş organizmalar, et ve süt
üretimi, gübreleme, akıllı tarım uygulamaları ve toprak profilleri gibi bölümleri içeren
farklı yaklaşımlarla tarıma yeniden bakış isimli kitabımızda, tarımın farklı konularında
birbirinden değerli bilim insanlarının yazdığı, tarımın mevcut durumunu çözüm önerileriyle
ortaya koyan bu eserin ülkemiz tarımına katkılar sağlaması temennimizdir.
Arı Yetiştiriciliği Sorunları ve Çözüm Önerileri
Genetic modification has great advantages in improving performance of bacteria, but its oral safety has not been systematically evaluated. In this study, the toxicity including the reproductive toxicity of two genetically modified bacteria engineered using food-grade vectors on three generations of rats (F0, F1 and F2)were studied. Sprague Dawley rats were administrated by gavage with corresponding parent and genetically modified Lactobacillus plantarum and Lactobacillus delbrueckii at 2.5 × 10 9 CFU every other day for 8 weeks. Results showed that the transgenic Lactobacillus had no significant toxicity to the body weight, food intake or blood biochemical parameters of three generations of rats. There was no significant effect on the reproductive parameters (gestational weight and pregnancy time) and related hormones (FSH, LH, estradiol, progesterone and testosterone) of the parent rats (F0 and F1). Consistently, their offsprings had no abnormal physical conditions including body weight, body length and anogenital distances. Moreover, their pups had normal body organ weight and reasonable abnormal sperm rate. Further analyses were conducted to evaluate SOD, MDA and GPX of the ovarium in the F1 and F2 female rats, which showed no malady phenotypes. In conclusion, transgenic Lactobacillus has no toxicity to the three generations of rats.
Herein, a handheld device for on-site detection of genetically modified crop was developed. To meet the requirement of highly sensitive detection, single-wavelength light emitting diode was used as excitation light source, silicon photodiode with a dark current of pA level was adopted for photoelectric conversion, and phase-sensitive signal acquisition and amplification were carried out to improve the signal-to-noise ratio. Specially, micron-scale supramolecular fluorescence material was proposed for the first time as the signal label of sandwich immunoassay, which is a luminescent molecule aggregate. It could bring much greater increment in fluorescence intensity by increasing the number of luminescent molecules and accelerating the internal energy recombination, resulting in an excellent detection sensitivity. Under optimal experimental conditions, genetically modified protein PAT/bar as a template could be detected in the range of 0.01 ~ 10000 ng/mL, with a limit of quantification10 pg/mL. Selectivity, stability, precision and accuracy were all satisfied. And, real GM rice sample could be successfully detected, implying that this high-sensitivity on-site detection platform has broad application prospects in food inspection.
A novel fluorescent biosensor was proposed for detecting the CaMV 35S promoter in genetically modified organisms (GMOs). It was based on a proximity extension mediated multiple cascade strand displacement amplification connected with CRISPR/Cpf 1 (termed PE-MC/SDA-CRISPR/Cpf1). In this protocol, the CaMV 35S was recognized by proximity reaction in the presence of two adjacent primer probes. The proximity extension further triggered the multiple cascade strand displacement amplification (MC/SDA), generating a mass of ssDNA. The products compelled the trans-cleavage activity of CRISPR/Cpf 1, so as to cleave nearby ssDNA-FQ reporters and generate a strong fluorescent signal. The ingenious three-link combination design allowed the CaMV 35S a low background interference. And the MC/SDA combined with CRISPR/Cpf 1 dramatically improved the detection sensitivity. Under optimized conditions, the detection linear range of ultrasensitive fluorescent biosensor for CaMV 35S was from 50 fM to10 pM and 10 pM–500 pM, along with the limit of detection (LOD) down to 14.4 fM. The sensing platform also had excellent performance in the assay of selectivity and real samples. Therefore, the method earned great application potential for transgenic crops.
Au nanoparticles (AuNPs) have been used as signal reporters in colorimetric lateral flow immunoassays (LFAs) for decades. However, it remains a major challenge to significantly improve the detection sensitivity of traditional LFAs due to the low brightness of AuNPs. As an alternative approach, we overcome this problem by utilizing 150 nm gold nanoshells (AuNSs) that were engineered by coating low-density silica nanoparticles with a thin layer of gold. AuNSs are dark green, have 14 times larger surface area, and are approximately 35 times brighter compared to AuNPs. In this study, we used detection of thyroid-stimulating hormone (TSH) in a proof-of-concept assay. The limit of detection (LOD) with AuNS-based LFA was 0.16 µIU/mL, which is 26 times more sensitive than the conventional colorimetric LFA that utilizes AuNP as a label. The dynamic range of the calibration curve was 0.16–9.5 µIU/mL, making it possible to diagnose both hyperthyroidism (<0.5 µIU/mL) and hypothyroidism (>5 µIU/mL) using AuNS-based LFA. Thus, the developed device has a strong potential for early screening and diagnosis of diseases related to the thyroid hormone.
Extensive safety testing is an impediment in the food industry, in particular, with its traditionally low margins of profit. The existing regulatory framework outlined in connection with the evaluation of the safety of foods seems to be sufficient to cope with genetically engineered organisms and their products. The safety issue of whole foods derived from genetically modified microorganisms, animals, and plants is more difficult than that of single food constituents or defined chemical mixtures that will be used in food. The examination of biological risks related to animal cell cultures and the consideration of the type of manipulation allows the determination of adequate containment level to protect human and environmental health. Food safety evaluation of transgenic animal is handled by national and international authorities. Plant cell cultures in bioreactors offer tremendous opportunities to produce a wide range of food ingredients or additives such as pigments, amino acids, flavors, and enzymes.
Near-infrared spectroscopy (NIRS) has become a more popular approach for quantitative and qualitative analysis of feeds, foods and medicine in conjunction with an arsenal of chemometric tools. This was the foundation for the increased importance of NIRS in other fields, like genetics and transgenic monitoring. A considerable number of studies have utilized NIRS for the effective identification and discrimination of plants and foods, especially for the identification of genetically modified crops. Few previous reviews have elaborated on the applications of NIRS in agriculture and food, but there is no comprehensive review that compares the use of NIRS in the detection of genetically modified organisms (GMOs). This is particularly important because, in comparison to previous technologies such as PCR and ELISA, NIRS offers several advantages, such as speed (eliminat-ing time-consuming procedures), non-destructive/non-invasive analysis, and is inexpensive in terms of cost and maintenance. More importantly, this technique has the potential to measure multiple quality components in GMOs with reliable accuracy. In this review, we brief about the fundamentals and versatile applications of NIRS for the effective identification of GMOs in the agricultural and food systems.
The fate of transgenic DNA (tDNA) and protein from feed derived from Genetically Modified organisms (GMOs) in animals has been a major issue since their commercialization in 1996. Several studies have investigated the risks of horizontal gene transfer (HGT) of tDNA and protein to bacteria or animal cells/tissues, but some of the reported data are controversial. Previous reports showed that tDNA fragments or proteins derived from GM plants could not be detected in tissues, fluids, or edible products from livestock. Other researchers have shown that there is a possibility of small fragments entering animal tissues, fluids and organs. This motivated us to update our knowledge about these concerns. Therefore, this review aimed to evaluate the probable transfer and accumulation of tDNA/proteins from transgenic feeds in animal samples (ruminant and non-ruminant) by evaluating the available experimental studies published scientifically. This study found that the tDNA/protein is not completely degraded during feed processing and digestion in Gastro-Intestinal Tract (GIT). In large ruminants (cattle), tDNA fragments/proteins were detected in GIT digesta, rumen fluid, and faeces. In small ruminants (goats), traces of tDNA/proteins were detected in GIT digesta, blood, milk, liver, kidney, heart and muscle. In pigs, they were detected in blood, spleen, liver, kidney, and GIT digesta. In poultry, traces were detected in blood, liver and GIT digesta but not in meat and eggs. Notwithstanding some studies that have shown transfer of tDNA/protein fragments in animal samples, we cannot rely on these few studies to give general evidence for transfer into tissues/fluids and organs of farm animals. However, this study clearly shows that transfer is possible. Therefore, intensive and authentic research should be conducted on GM plants before they are approved for commercial use, investigating issues such as the fate of tDNA or proteins and the effects of feeding GM feed to livestock.
This complete and well-organized overview of chemiluminescence and bioluminescence is divided into two parts. The first covers historical developments and the fundamental principles of these phenomena before going on to review recent advances and instrumentation. The second part deals with the applications in a variety of research fields including life sciences, drug discovery, diagnostics, environment, agrofood, and forensics. The book is suitable not only for researchers currently employing detection techniques in their research activity, but also for those approaching the subject for the first time. Particular emphasis is placed on the use of chemiluminescence and bioluminescence for the development of a variety of (bio)analytical methods, such as flow-assisted methods, enzyme-, antibody- or gene probe-based assays also in multiplexed formats, miniaturized analytical devices, biosensors, BRET and protein complementation assays, whole-cell biosensors, and bioluminescence molecular imaging. Individual chapters are devoted to the most important and rapidly developing fields including: Instrumentation for Chemiluminescence and Bioluminescence; In vivo Molecular Imaging; Biotechnological Improvements of Bioluminescent Systems; Cell-based Bioluminescent Biosensors, and Miniaturized Analytical Devices Based on Chemiluminescence, Bioluminescence and Electrochemiluminescence. The book also includes a comprehensive collection of recent bibliographic references.
With the advancement of science and technology, there is an urge to put less effort into our activities be it some of our household works, official work, or any computational work. These upgrading techniques have one thing in common is to save time. Similarly, with the progress of new discoveries and inventions in any field of science, the quest to portrait the ancestral roots of the phylogenetic tree drew the researchers’ attention to the rapid and real-time assessment of species detection. The classic species identification and classification system mainly includes the systematic positioning of an individual according to their morphology, appearance, and other phenotypic characteristics. Although species identification and authentication processes need some rapid technique that can conserve time as well as work on a scientific basis and with efficiency.
Lateral-flow assays (LFAs) are quick, simple and cheap assays to analyze various samples at the point of care or in the field, making them one of the most widespread biosensors currently available. They have been successfully employed for the detection of a myriad of different targets (ranging from atoms up to whole cells) in all type of samples (including water, blood, foodstuff and environmental samples). Their operation relies on the capillary flow of the sample throughout a series of sequential pads, each with different functionalities aiming to generate a signal to indicate the absence/ presence (and, in some cases, the concentration) of the analyte of interest. To have a user-friendly operation, their development requires the optimization of multiple, interconnected parameters that may overwhelm new developers. In this tutorial, we provide the readers with: (i) the basic knowledge to understand the principles governing an LFA and to take informed decisions during lateral flow strip design and fabrication, (ii) a roadmap for optimal LFA development independent of the specific application, (iii) a step-by-step example procedure for the assembly and operation of an LF strip for the detection of human IgG and (iv) an extensive troubleshooting section addressing the most frequent issues in designing, assembling and using LFAs. By changing only the receptors, the provided example procedure can easily be adapted for cost-efficient detection of a broad variety of targets.
Increased Genetically Modified (GM) plant production and the widespread trade and use of Genetically Modified Organisms (GMOs) in the food and animal feed markets are questioned about food safety by consumers. GMOs have been the subject of various cases in the areas of public health, environment, and finance. Turkey has also regulations and serious penalties about GMOs and its product-usage so it is also questioned by forensic sciences. The purpose of this study is to investigate the current situation by making GMO analyses in risky product groups. Products containing corn and soy content, known as risky product groups, were obtained from markets in Istanbul. GMO screening analysis of 35 products selected between July-September 2018 was performed by Real-Time PCR method. Positive results were detected in 2 animal feed samples. In these samples, the amount of GMO (35S region) was determined as below 0.1%. According to the legal regulations, GMO below 0.9% rates may result from contamination that cannot be prevented. Öz: Genetiği değiştirilmiş (GD) bitki üretim ve ekiminin artması, genetiği değiştirilmiş organizmaların (GDO) gıda ve yem pazarında yaygın ticareti ve kullanımı gıda güvenliği konusunda tüketiciler tarafından sorgulanmaktadır. GDO'lar halk sağlığı, çevre ve finans alanlarında çeşitli davaların konusu olmuştur. Türkiye, pek çok ülke gibi GDO'lar ve ürünlerine dair hukuki düzenlemelere ve ciddi yaptırımlara sahiptir. Bu sebeple adli bilimler açısından pek çok açıdan önemli bir konu olmaktadır. Bu çalışmanın amacı riskli ürün gruplarında GDO analizleri yaparak şu anki durumu araştırmaktır. Riskli ürün grupları olarak bilinen mısır ve soya içeriği barındıran ürünler İstanbuldaki marketlerden temin edilmiştir. Temmuz-Eylül 2018 tarihleri arasında seçilen 35 adet ürünün, Real-Time PCR yöntemi ile GDO tarama analizi yapılmıştır. İki hayvan yem örneğinde pozitiflik tespit edilmiştir. Bu örneklerde GDO miktarı (35S bölgesinde) %0.1'den düşük olarak tespit edilmiştir. Yasal düzenlemelere göre %0.9'un altında bulunan GDO oranlarının ise önlenemeyecek kontaminasyonlardan kaynaklanabileceği kabul edilmektedir. Anahtar kelimeler: Biyoteknoloji, genetik, polimeraz zincir reaksiyonu (PCR), halk sağlığı.
Microbes are ubiquitously present in nature and their presence in environment or food materials is natural and unavoidable. Presence of microbes in food products may be beneficial or may cause serious threat to health upon consumption. With reference to European Commission (EC), Food and Drug Administration (FDA) and Hazard Analysis Critical Control Point (HACCP) guidelines, detection of illness causing foodborne microbes/pathogens and/or presence of GMOs has become a burning issue that needs to be attended on priority basis. This chapter is an attempt to describe various molecular techniques for detection of foodborne pathogens and GMOs. Emphasis has been made to elaborate basic to next-generation techniques developed related to PCR, microarrays, and DNA sequencing that have achieved unprecedented level of performance in terms of sensitivity, speed, precision, and accuracy.
Among the existing multiplex genetically modified organism (GMO) detection methods, significant problems are highlighted, including amplification asymmetry of different targets, and the low detection throughput, which limits their capacity to meet the requirements of high-throughput analysis. To mitigate these challenges, a ‘turn-on’ ultra-sensitive multiplex real-time fluorescent quantitative biosensor is developed. In this system, the multiplex ligation-dependent amplification (MLPA), universal primer and universal probe are innovatively combined, which can enhanced the amplification specificity, overcome asymmetric amplification and guarantee the homogeneity of amplification efficiency simultaneously. Furthermore, both single and multiplex detection results can be output by the fluorescent group labeled on universal TaqMan probes for different targets in real-time. After optimization, the quantitative detection limit was 5 pg. In conclusion, this strategy could serve as an important tool for GMO detection in processed and commercially available products, even in the fields that require reliable and sensitive detection of DNA targets.
Gold nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective post-hybridization manipulations for genomic analysis. Here, we present a novel design for a colorimetric gene-sensing platform based on the CRISPR/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the trans-ssDNA or -ssRNA cleavage. Target-induced trans-ssDNA or ssRNA cleavage triggers an aggregation behavior change for the designed AuNPs-DNA probes pair, enabling the completion of naked-eye gene detection (transgenic rice, African swine fever virus, and miRNAs as the models) within 1 hour. This platform is also showing promise as a fast and inexpensive tool for bacteria identification using 16S ribosomal DNA or 16S ribosomal RNA. CRISPR/Cas-based colorimetric platform shows superior characteristics, such as probe universality, compatibility with isothermal reaction conditions, on-site detection capability, and high sensitivity; thus represents a robust next-generation gene detection platform.
In this work, the interactions between adenine–adenine di-nucleotide (DA2N) and carbon nanotube (CNT) in the presence of Lysyllysine (LL) was studied by the molecular dynamics simulation. Different carbon nanotubes including (5.5), (6.6) and (7.7) were used to investigate the effect of CNT type. The binding energies were calculated using the molecular mechanics-Poisson Bolzmann surface area method. The results showed that the contribution of the van der Waals interactions between DA2N and CNT was greater than that of the electrostatic interactions. The LL significantly enhanced the electrostatic interactions between the DA2N and CNT (6.6). The quantum calculations revealed that the sensor properties of the DA2N were not significantly affected by the CNT and LL. However, the five-membered ring of adenine played a more important role in the sensing properties of the DA2N. The obtained results are consistent with the previous experimental observations that can help to understand the molecular mechanism of the interaction of DA2N with CNT.
Graphic abstract
Open image in new window
Genetic transformation in plants agreements a great potential to modify crops for improved agronomic traits including resistance to diseases, pests and good nutritional quality along with enhanced productivity. The transgene could be derived from unrelated plant species and even from non-plant sources leading to a revolution in molecular agriculture. In this chapter, the main approach lies on concept of genetic engineering techniques to improve the plant architect. The concept of GM crops and environmental implications besides their safety assessment is documented in detail and also in the end future perspective for adopting the next generation quantitative genetics is also elaborated.
Background
Legislation regulating for labeling and use of genetically modified (GM) crops are increased considerably worldwide in order to health and safety assurance of consumers. For this purpose, a polymerase chain reaction (PCR) method has been developed for detection of GM rice in people’s food diet.
Methods
In this study, eighty-one non-labeled rice samples were collected randomly from different market sites of Tehran, Iran. In order to analysis, rice genomic DNA was extracted using MBST DNA extraction kit and subsequently, sucrose phosphate synthase (SPS) gene was used to confirm the quality of extracted DNA. Then, cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium nopaline synthase (NOS) terminator were selected as screening targets for detection of GM rice sequences by PCR.
Results
According to our results, 2 out of 81 (2.4%) samples tested were positive for CaMV 35S promoter while no positive result was detected for NOS terminator.
Conclusion
The obtained data indicated that this method is capable to identify the GM rice varieties. Furthermore, it can demonstrate the possibility of the presence of GM rice in Tehran’s market, thus putting emphasis on the requirement for developing a precise approach to evaluate this product.
A new polyclonal antibody that recognizes the CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS), which provides resistance to glyphosate in soybean (Roundup Ready®, RR soybean), was produced. New Zealand rabbits were injected with a synthetic peptide (Pc_312-324, (PEP)) present in the soybean CP4-EPSPS protein. The anti-PEP antibodies production was evaluated by electrophoresis (SDS-PAGE) and an enzyme-linked immunosorbent assay (ELISA) was developed in order to study their specificity. The ELISA showed that the polyclonal antibody was specific to PEP. In addition, the anti- PEP was immobilized onto a gold disk electrode and the antigen-antibody interaction was evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Moreover, the EIS showed that the electron transfer resistance of the modified electrode increased after incubation with solutions containing CP4-EPSPS protein from RR transgenic soybean, while no changes were detected after incubation with no-RR soybean proteins. These results suggest that the CP4-EPSPS was immobilized onto the electrode, due to the specific interaction with the anti-PEP. These results show that this antigen-antibody interaction can be detected by electrochemical techniques, suggesting that the anti-PEP produced can be used in electrochemical immunosensors development to quantify transgenic soybean.
Crops undergo artificially DNA modifications for improvements are considered as genetically modified (GM) crops. These modifications could be in indigenous DNA or by introduction of foreign DNA as transgenes. There are 29 different crops and fruit trees in 42 countries, which have been successfully modified for various traits like herbicide tolerance, insect/pest resistance, disease resistance and quality improvement. GM crops are grown worldwide and its area is significantly increasing every year. Many countries have very strict rules and regulations for GM crops and are also a trade barrier in some situations. Hence, identification and testing of crops for GM contents is important for identity and legitimacy of transgene to simplify the international trade. Normally, molecular identification is performed at three different levels, i.e., DNA, RNA and protein, and each level has its own importance in testing about the nature and type of GM crops. In this chapter, current scenario of GM crops and different molecular testing tools are described in brief.
İzlenebilirlik; üretim, işleme ve dağıtımın tüm aşamaları boyunca bitkisel ve hayvansal ürünlerin, gıda ve yemin, gıdanın elde edildiği hayvanın veya bitkinin, gıda ve yemde bulunması amaçlanan veya beklenen bir maddenin izinin sürülebilmesi ve takip edilebilmesidir. İzlenebilirlik üretim ve dağıtım aşamaları, ithalat da dâhil olmak üzere birincil üretimden nihai tüketiciye satışa kadar olan aşamaların tümünü kapsar ve ilgili gıdada insan sağlığını en yüksek düzeyde korumayı amaçlar. İzlenebilirlik sistemi tüm ürün ve girdilerin, birim veya partilerinin tanımlanmasını; bunların nereden, ne zaman ve nereye hareket ettiklerine ilişkin bilginin toplanması ve saklanmasını ve bu iki veriyi birbiri ile ilişkilendirecek bir sistemin kurulması aşamalarını içermektedir. İzlenebilirlik resmi kontroller açısından olduğu kadar, uluslararası gıda ticaretinin de yönlendirici Gıda Güvenliği Yönetim Standartları olan BRC ve IFS gibi uluslararası belgelendirme faaliyetleri ve ülkemizdeki gıda ticaretinin sağlıklı işlemesi açısından da kritik öneme sahiptir. Gıda güvenliği ve kalitesini önemli ölçüde garanti altına alan izlenebilirlik sistemleri, son yıllarda işletmeler ve düzenleyiciler için önemli yer tutmaktadır. İzlenebilirlik sistemleri, hammaddenin türüne, ürün yelpazesine, şartnameye ve işletmenin teknolojik olanaklarına göre değişmektedir.
Los organismos genéticamente modificados (OGM) y en particular los cultivos genéticamente modificados (GM), son el resultado de la modificación de la información genética de una especie a partir del uso de la biotecnología moderna para proporcionar nuevas características que su contraparte no modificada no posee, tales como resistencia a insectos, tolerancia a herbicidas, contenido de nutrientes entre otros. La mayor parte de estos cultivos se concentran en cuatro productos: soya (Glycine max), maíz (Zea Mays), canola (Brassica napus) y algodón (Gossypium hirsutum); y los principales productores son Estados Unidos, Brasil, Argentina, India y Canadá. Por su parte, Colombia ocupa el puesto 18 con cultivos de maíz, algodón y claveles azules. La introducción de estas especies en cualquier mercado está limitada por la legislación propia del país destino, así como por los estudios que permiten establecer su efecto sobre el medio ambiente, la salud humana y animal; en este sentido, la precisión y confianza de las técnicas analíticas empleadas en la evaluación del contenido de OGM son un elemento importante para la toma de decisiones basadas en evidencias objetivas, especialmente frente al debate en torno a su uso. Este documento presenta una revisión de las tecnologías de análisis más importantes disponibles a nivel mundial, frente a las capacidades nacionales para su detección.
In this study twenty six maize kernels and 64 processed maize food including maize flour, starch, corn flakes were collected from different markets located in Turkey, analyzed for genetic modification using the polymerase chain reaction. The samples were examined for the presence of genetic elements located in the majority of transgenic crops such as NOS terminator and CaMV 35S promoter using conventional PCR and then verified real time PCR, too. Additionally, Bt11, Bt176, Mon810, CBH351 and T25 events which have been enjoyed, limited data in Turkey, examined in the products using conventional PCR. Then, quantification of the all lines (except CBH351) was performed via real time PCR too. The results indicated that foreign genetic elements were found in analyzed 14 samples raw and processed materials and the quantity in one sample (maize flour) was greater than 0.9%, the limit at which labeling is required by Biosafety Law in Turkey.
Genetic modification (GM) techniques have been an important research area of food and feed industry since the 19th century. There is a strong consumer concern over genetically modified organisms (GMOs) because of their potential risks on health and environment. For this purpose, various countries including Turkey have released labelling regulations for products derived from GMOs. These legal enforcements brought the necessity for reliable detection methods. The aim of our study was to evaluate the effect of processing on the detection possibility of GMOs by using a commercial Enzyme Linked Immunosorbent (ELISA) assay. For this, flour mixtures containing 0.5%, 1%, 5%, 10%, 100% were prepared by mixing the appropriate amount of RUR-GM and non GM standard soy flour and main processing techniques most used in the food industry (baking, autoclaving and freezing) were applied. According to our results, the detection of GMOs was possible at all concentrations of autoclaved and frozen samples. In dry heated samples, GMOs could not be detected containing below 5% GMOs. ELISA method cannot be recommended as a reference method for evaluation of the compliance with the regulations, but it can serve as a practical alternative to be used as an online monitoring tool in production lines for raw and mildly processed foods.
Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.
In response to growing concerns regarding the use of genetically modified food in Europe, revisions have been proposed to
Directive 90/220 of the European Economic Community. The amendments would provide more specific guidance for risk assessment
and monitoring as a way of reassuring the public about the safety of products introduced on the market. There is a clear need
for the United States to harmonize with this approach if international trade is not to be impeded.
Current market conditions and food regulations make it necessary for international and domestic participants in the agrifood industry to structure supply chains that control the content of genetically modified (GM) material in their products. Tests to detect and/or quantify GM components represent an important tool in maintaining such supply systems. This study assesses the field performance of kits that employ lateral flow immuno-technology to detect soybeans GM to be resistant to the herbicide glyphosate. Operators at 23 grain-handling facilities were paid to conduct analyses on a series of blinded samples containing defined proportions of conventional and transgenic soybeans. The observed rate of false positives was 6.7% in an experiment in which the highest level of GM material was 1% and 22.3% in a second experiment in which the highest level of GM material was 10%. This difference may be attributed to increased risk of cross-contamination with the higher level of transgenic material used in the second study. Samples containing 0.01% GM material were reported as genetically modified 6.70% of the time, while samples containing 0.1, 0.5 and 1% GM material were classified as genetically modified 29.5, 67.7 and 68.2% of the time, respectively. Thus, the frequencies of false negatives were 93.3, 70.5, 33.3 and 31.8% for samples containing 0.01, 0.1, 0.5 and 1.0% GM material. Samples containing 10% GM material were correctly reported as genetically modified in all cases. These results lead to the conclusion that the kit under study is useful in screening for lots of soybeans that contain high levels of GM material, but that, as a field tool, it is not effective in monitoring for GM material at the level of 1.0% or lower. Statistical and immunochemical analyses were carried out in order to assess the relative contributions of various factors to the error observed in these studies. These analyses indicated that limitations in operator performance, not defects in test kit materials, were the primary contributors, while sample size may play a secondary role. As both operator performance and sample size are independent of the specific characteristics of the test kit used in this study, it appears justifiable to generalize conclusions obtained here to other similar test systems.
We investigated random amplified polymorphic DNA (RAPD) in 27 inbred barley lines with varying amounts of common ancestry and in 20 doubled-haploid (DH) lines from a biparental cross. Of 33 arbitrary 10 base primers that were tested, 19 distinguished a total of 31 polymorphisms. All polymorphisms were scored as dominant genetic markers except for 1, where Southern analysis indicated the presence of two codominant amplification products. The inheritance of 19 RAPD polymorphisms and one morphological trait was studied in the DH lines. There was no evidence for segregation distortion, but a group of four tightly linked loci was detected. The frequencies of RAPD polymorphism in pairs of inbred lines were used to compute values of genetic distance (d), which were compared to kinship coefficients (r) between the same pairs of lines. A linear relationship between r and d was evident, but low values of r gave poor predictions of d. Cluster analysis showed that groups of inbred lines based on r were similar to those based on d with some notable exceptions. RAPD markers can be used to gain information about genetic similarities or differences that are not evident from pedigree information.
DNA-based analytical methods are often used to verify the presence of genetically modified organisms (GMOs) in food. In Switzerland,
a preliminary study, organized by a subcommission of the Swiss Food Manual, of different polymerase chain reaction (PCR) systems
for the detection of GMOs showed that the application of qualitative PCR systems can lead to interlaboratory differences of
at least a factor of 10. These differences can be diminished using internal standards (competitors). The quantitative competitive
(QC) PCR for the detection of the 35S promoter or the NOS terminator in food samples is presented. The GMO content of food
samples can be determined using QC-PCR.
We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.
We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.
The analysis of differential gene expression has become increasingly important in recent years. Typically, differentially expressed genes are identified in a primary screening procedure, yielding candidate genes whose differential expression has to be verified. We provide a highly sensitive, efficient and nonradioactive differential screening procedure to analyze numerous candidate genes in a single step. This comprises labeling of poly(A)+ RNA of the cell types analyzed with DIG Chem-Link and differential hybridization to the candidate genes fixed on dot blots. DIG Chem-Link allows, to our knowledge, for the first time efficient and direct nonradioactive labeling of RNA in vitro. Advantages of this method include extremely short exposure times and the feasibility to re-use the probes after prolonged storage. Using this procedure, we isolated several genes that are differentially expressed in maturing Langerhans cells.
Recent controversies about genetically modified foods in the United Kingdom and several other European countries highlight the apparent differences that exist in public opinion on this subject across the Atlantic. Why are people in the United States seemingly untroubled by a technology that causes Europeans so many difficulties? The results of survey research on public perceptions of biotechnology in Europe and the United States during 1996-1997, together with an analysis of press coverage and policy formation from 1984 to 1996, can help to answer this question.
This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.
An immunoassay for detection of a specific genetically modified soybean (Roundup-Ready) was validated on dried soybean powder in an interlaboratory study. Different percentages of genetically modified soybeans in nonmodified soybean matrix were evaluated in a blind study. Thirty-eight laboratories from 13 countries participated. The immunoassay was evaluated for 2 endpoints: (1) To give a semiquantitative result, i.e., determination of a given sample above or below a given threshold, or (2) to compute a quantitative result, i.e., percentage of genetically modified soybeans in the sample. Semiquantitative results showed that a given sample which contained <2% genetically modified soybeans was identified as below 2% with a 99% confidence level. Quantitative use of the assay resulted in a repeatability (r) and reproducibility (R) that were computed to be RSDr = 7% and RSDR = 10%, respectively, for a sample containing 2% genetically modified soybeans. Application of this method depends on availability of appropriate reference materials for a specific food matrix. Only matrix-matched reference materials can be used for analysis of food or food fractions.
In this study, the application of a qualitative and a quantitative method of analysis to detect genetically modified RR-Soy (Roundup-Ready™ Soy) in processed foods is described. A total of 179 various products containing soy such as baby food and diet products, soy drinks and desserts, tofu and tofu products, soy based meat substitutes, soy protein, breads, flour, granules, cereals, noodles, soy bean sprouts, fats and oils as well as condiments were investigated following the pattern of the § 35 LMBG-method L 23.01.22-1. The DNA was extracted from the samples and analysed using a soybean specific lectin gene PCR as well as a PCR, specific for the genetic modification. Additional, by means of PCR in combination with fluorescence-detection (TaqMan® 5'-Nuclease Assay), suspicious samples were subjected to a real-time quantification of the percentage of genetically modified RR-Soy. The methods of analysis proved to be extremely sensitive and specific in regard to the food groups checked. The fats and oils, as well as the condiments were the exceptions in which amplifiable soy DNA could not be detected. The genetic modification of RR-Soy was detected in 34 samples. Eight of these samples contained more than 1 % of RR-Soy. It is necessary to determine the percentage of transgenic soy in order to assess whether genetically modified ingredients were deliberately added, or whether they were caused by technically unavoidable contamination (for example during transportation and processing).
The extent to which sampling of raw materials and foods, for detection of the presence of GMOs, presents a significant problem depends on the type of material to be sampled, the purpose of the analysis and the degree of risk that is acceptable in obtaining a wrong result. Sampling for indications of non-segregation or co-mingling of raw materials is the main area for which sample plans will for the future need to be developed. Fortunately, there is considerable experience in sampling of commodities in analogous areas such as for the presence of mycotoxins in cereals and nuts where well-tested sampling plans could be utilised.
Uncontrolled cell proliferation is the hallmark of cancer, and tumor cells typically have acquired damage to genes that directly regulate their cell cycles (4). Interplay between the products of the cyclin D1, p16(INKA4) and retinoblastoma (Rb) susceptibility genes is involved in the regulation of cell cycle progression from G1 to S. Cell progression from G1 to S requires, among other things, activation of specific kinases, Cdks (Cdk4 and Cdk6), in association with cyclin D1. The active Cdk/cyclin D1 complex phosphorylates sequentially the Rb protein, thus releasing Rb-bound transcription factors of the E2F family. Free E2Fs transactivate genes that are essential for entry into S and DNA replication (S-phase effectors). Cdk/cyclin D1 activity is negatively regulated by binding of several cyclin kinase inhibitors, including p16(INKA4). The p16(INKA4) is believed to be activated in response to growth control stimuli through pathways which are not fully elucidated. Because pRb, cyclin D1 and p16(INKA4) are all upstream regulators of E2f activity, the frequent involvement of these proteins in human cancer suggests a central role for E2F in control of cell proliferation (9). Genetic alterations affecting p16(INKA4) and cyclin D1 protein that govern phosphorylation of the Rb protein and control exit from the G1 phase of the cell cycle, are so frequent in human cancers that inactivation of this pathway may well be necessary for tumor development. Like the p53 protein, component of this Rb pathway, although not essential for the cell per se, may participate in checkpoint functions that regulate homeostatic tissue renewal through life (10). It was recently recognized that the regulation of cell death (apoptosis) is also an important modulator of tumorigenesis. At least two genes linked to human cancers, bcl-2 and TP53, have been shown to regulate apoptosis. Cell culture studies have demonstrated that TP53 can induce, and bxl-2 suppress apoptosis in response to various stimuli. This raises the question as to how dysregulation of apoptosis contributes to neoplastic transformation and malignant cell growth (11). Genes for all these markers have been identified, isolated and sequenced, making it possible to apply molecular-based technologies for cancer detection. The continued acceleration of development of scientific information and new techniques necessitates the need for clinical application. Research in molecular genetics, cell biology, protein chemistry and immunology has identified many early changes occurring during neoplastic progression such as novel proteins, growth factors, cytokines, DNA damage and multiple genetic alterations. These changes present in tissue and other body fluids (e.g., plasma, urine, sputum, lung washings and feces) are now recognized as markers for impending cancer or for risk of cancer development. To explore fully the application of molecular profiles for earlier detection and risk assessment, it is essential to understand the molecular pathogenesis of cancer (i.e., the natural history of tumor progression) so that the biological behavior of an evolving lesion can be predicted with greater accuracy. Risk assessment may involve testing of genetic markers at different loci or specific genes associated with cancer predisposition. Current findings indicate that cancers usually evolve through many complex cellular processes, pathways and networks. A better understanding of the circuits in these pathways is essential if we are to successfully apply these molecular-based technologies to early detection (15). Progress in the field has, however, been hampered by hurdles such as fragmentation and lack of coordination of systematic application of these molecular markers, and lack of research emphasis on the continuum of preclinical tumor development and on evaluation of new techniques and their clinical application. As a consequence, much work in this area has been fragmented into numerous small and disconnected studies and could not therefore be generalized to the general population as a whole (16). The application of emerging molecular technologies in early cancer detection and risk assessment is a high priority area in NCI strategy for fight against cancer. I have presented in this review several markers and promising molecular techniques that may facilitate cancer detection and risk assessment if studied, and have outlined several areas of research that need to be carried out.
Qualitative detection methods for genetically modified (GM) DNA sequences in foods have evolved fast during the past years. The sensitivity of these systems is extremely high, even for processed foodstuffs. However, in future, quantitative results about the fraction of GM material in a composite food will be needed and the fast increasing number of GM foods on the market demands the development of more advanced multi-detection systems. Other challenges and problems might arise from the decreasing relevance of methods which screen for sequences commonly found in GMOs, the inability to detect GM foods for which the modified sequence is unknown, the lengthy standardisation procedures and the need to up-date continuously databases comprising commercially available GM foods and the respective detection strategies.
The Cartagena Protocol on Biosafety was adopted in Montreal, Canada, in the early hours of the morning of 29 January 2000, following last-minute speculation that the negotiations were in danger of collapsing again, as they had in Colombia the previous year. The protocol, negotiated under the United Nations Convention on Biological Diversity,1 is one of the first legally binding international agreements to govern the transboundary transfer of genetically modified organisms (GMOs), primarily for use in agriculture. Given the intense and highly charged nature of the debate that has raged around the use of biotechnology in agriculture in recent years, [2 ] it was striking to hear all those present in Montreal, including governments, the private sector, and green groups, express their satisfaction with the outcome. What does this universal endorsement in such a highly contentious arena indicate? What does the protocol accomplish for different groups and for global governance of the safe use of biotechnology in ag riculture? Use of GMOs in agriculture has expanded at a rapid rate in key agricultural exporting countries in recent years. Countries where transgenic crops are in advanced stages of field testing or commercialization include the United States, Argentina, Canada, and Australia, with Spain, France, Mexico, South Africa, and China contributing a small percentage. The global area devoted to transgenic crops has increased dramatically in the last half-Decade--from 1,7 hectares in 1996 to 27.8 hectares in 1998. [3] National regulatory frameworks for safe use of biotechnology date from the early 1970s in the United States with countries such as Denmark, the United Kingdom, and Germany following suit in the 1980. [4] The most comprehensive cross-national regulations in this realm are the European Union (EU) regional directives covering contained use and deliberate release of GMOs, adopted in 1990. [5] Most developing countries have only recently begun to develop domestic biosafety regulations, although some, including India and Brazil, have regulations dating back to the early to mid-1990s. [6] Although a few existing international conventions cover different aspects of the trade in GMOs and voluntary guidelines have been developed by a number of international agencies for safe use of biotechnology, [7] the Cartagena Protocol is the first to mandate the need for consent of an importing country prior to trade in some GMOs, to allow for assessment of potential risks posed by such transfers to biodiversity and human health in the importing country (key provisons of the adopted protocol are described in the box on page 25). [8] Although originally demanded by developing countries and not seen as necessary by most Organisation for Economic Cooperation and Development (OECD) countries, the biosafety protocol negotiations have unfolded over a four-year period of expanding awareness and public concern, especially in Europe, over ecological and food safety concerns relating to living modified organism (LMO) use in agriculture and a concurrent escalating trade conflict between the United States and the European Union in this area, A de facto moratorium has been in effect against entry of transgenic crops into Europe over the last two years, as the European Community has debated amendments to its directives and has halted new approvals until such amendments are in place. This has transformed what began largely as a developed versus developing world issue into a growing intra-OECD conflict, with important repercussions for the nature of the finalized protocol and its relevance for different groups. [9] The Rocky Road to Cartagena The first suggestion that there was a need for a provision on biosafety under the Convention on Biological Diversity came in the early 1990s from the Malaysian delegation during negotiation of the convention. The 1986 testing of a genetically modified rabies vaccine in Argentina by a United States research institute without the knowledge of the Argentine government, led to concerns that developing countries would become testing grounds for novel and potentially risky substances that they had neither the capacity nor the regulatory frameworks in place to deal with. [10] Therefore, these countries called for informed consent prior to LMO transfers, as the recently concluded Basel Convention did for trade in hazardous waste to prevent dumping in developing countries. [11] Although this developing country demand drew sustained opposition from the United States, Japan, and biotechnology industry groups and only lukewarm support from the European Union, it was strongly supported by green groups and a number of key Nordic countries such as Denmark, and it proved difficult to dismiss. [12] The result was Article 19.3 of the Convention on Biological Diversity, which calls on parties to the convention to assess the need for and modalities of a protocol on biosafety. [13] After much protracted debate, the conference of the parties to the Convention on Biological Diversity made a decision in Jakarta in November 1995 to establish an ad hoc Working Group on Biosafety (BSWG) to negotiate a protocol. When negotiations finally commenced in 1996, a bewildering array of concerns faced countries trying to determine what a protocol on biosafety should cover. Thus, the first four BSWG meetings, which occurred between 1996 and 1998, were largely prenegotiation sessions, as countries, especially developing countries, sought to clarify their interests and determine their priorities for what to include within a protocol on biosafety.
Microarray technology is becoming an important comprehensive tool to study gene expression in plants. However, the use of this technology is limited by the large amount of sample tissue needed for microarray analysis. Generally, 50–200 µg of total RNA and 1–2 µg of mRNA is required for each hybridisation, which is equivalent to 50–100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of microarrays for transcript profiling in specific tissues and cell types during plant development. Here we report on a robust and reliable target amplification method that enables transcript profiling from sub-mg amounts of plant tissue. Using 0.1 µg of total RNA we show that twofold expression differences are possible to distinguish with 99% confidence. We also demonstrate the application of this method in an analysis of secondary phloem development in hybrid aspen using defined tissue sections, corresponding to 2–4 cell layers with a fresh weight of ∼0.5 mg.
The surveillance of food labelling concerning genetically modified organisms (GMOs) requires DNA-based analytical techniques.
Present assay systems allow the detection of GMO in food; however, they do not permit their quantitation. In this study, we
report the development of quantitative competitive polymerase chain reaction (QC-PCR) systems for the detection and quantitation
of the Roundup Ready soybean (RRS) and the Maximizer maize (MM) in food samples. Three DNA fragments that differ from the
GMO-specific sequences by an insertion were constructed and used as internal standards in the PCR. These standards were calibrated
by co-amplifying with mixtures containing RRS DNA and MM DNA, respectively. The calibrated QC-PCR systems were applied to
nine commercial food samples containing RRS DNA and to three certified RRS flour mixtures in order to elucidate whether these
food samples contain more or less than 1% RRS DNA. Finally, the GMO contents of four samples that were found to contain more
than 1% RRS were determined by QC-PCR using various amounts of standard DNA.
Protoplast-derived, transformed maize plants were evaluated by Southern analysis for the presence of the aph IV gene which codes for resistance to the antibiotic, hygromycin B. This gene was used as a selectable marker for the transformation
of maize protoplasts. Southern analysis was performed with fluorescein-labeled probe DNA. A new method for labeling molecular
weight markers with fluorescein-N6 is presented. The nonradioactive Southern analysis method is compared to the radioactive method and the results show that
the nonradioactive method is as sensitive as the radioactive method.
The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products.Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil.If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker.Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity.Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers.
Immunoassay methodology is currently the method of choice for the quantitative and semi-quantitative detection of many types of proteins in complex mixtures. A combination of sensitivity, specificity and cost-effectiveness in terms of analytical performance are allied to a diverse array of assay formats suitable for laboratory and field use. In the present article, the problems of setting up immunoassays for novel food proteins, and the questions that need to be answered if a successful outcome is to be achieved, are discussed. Speculation on future developments in immunochemistry leads to the conclusion that antibody technology will play an important role in detection of novel proteins from genetically modified organisms.
We have developed immunological-based detection methods to support labeling of protein-containing food fractions derived from Roundup Ready® soybeans. Western blotting and enzyme linked immunosorbent assay (ELISA) procedures were developed to measure the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein derived from the Agrobacterium sp. strain CP4 in the major processed fractions derived from Roundup Ready soybean. Expression of the CP4 EPSPS protein confers tolerance to Roundup® herbicide. The western blotting method utilizes a polyclonal goat anti-CP4 EPSPS antibody which specifically binds to CP4 EPSPS followed by detection of bound goat antibody with biotinylated Protein-G. Detection of this complex is accomplished using horseradish-peroxidase (HRP) labeled NeutrAvidin™ and signal development by enhanced chemiluminesence. Data from western blot analyses of these fractions establish that stable epitopes remain after the seed has been subjected to processing conditions typically employed by the food industry, thereby enabling development of an ELISA method. The ELISA for measurement of CP4 EPSPS is a triple antibody sandwich procedure utilizing a monoclonal capture antibody and a polyclonal detection antibody followed by a third biotin labeled monoclonal anti-rabbit antibody. Sandwich formation is detected using HRP labeled NeutrAvidin™ with color development using TMB substrate. In the sandwich ELISA, the immunological activity of CP4 EPSPS was reduced by the extraction method required to solubilize CP4 EPSPS protein from processed fractions. Sensitivity of the CP4 EPSPS ELISA was sufficient to detect CP4 EPSPS protein in processed soybean fractions that contained 2% Roundup Ready soybean mixed with conventional processed soybean fractions, thereby making the ELISA an acceptable method to assess CP4 EPSPS protein in processed soybean fractions. Data on sensitivity, accuracy, precision and specificity, established that the western blot and ELISA methods are appropriate for compliance with the EC Novel Foods Regulation.
The capability of immunoassays to quantitatively detect proteins, be implemented commercially on a wide scale and generate validated data for regulatory compliance in a cost-effective manner is evidenced by the existing immunoassay industry. The use of immunoassays in the food industry is well established. To enable immunoassay testing of foods for the presence of genetically modified organisms (GMO), proprietary proteins must be available for test development. Standard reference materials are required for method development, to translate test results in terms of % GMO, and to ensure uniform test performance throughout the EU. Data are presented demonstrating quantitative detection of Roundup Ready® Soy Bean Certified Reference Material using a commercial ELISA. Strategies for successful implementation of testing are discussed.
Present polymerase chain reaction (PCR) detection methods only allow the qualitative detection of GMO in food without quantitation of the GMO content. Clearly, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. This quantitative competitive (QC) PCR was first described in the early nineties and is widely used nowadays.We have developed and evaluated QC–PCR systems for the quantitative detection of Roundup ReadyTM soybean (RRS) and Maximizer maize (MM) in food samples. Three DNA fragments differing from the GMO specific sequences by DNA insertions were constructed and used as internal standards in QC–PCR. These standards were calibrated by co-amplifying with mixtures containing defined amounts of RRS DNA and MM DNA, respectively. The calibrated QC–PCR systems were applied to several commercial food samples containing RRS and to three certified RRS flour mixtures (Fluka standards). Recently, quantitative methods for the detection of RRS were successfully tested in a collaborative study involving twelve European control laboratories. Thus, QC–PCR methods will allow to survey “de minimis thresholds” of GMOs in food.
Due to the market introduction of genetically modified crops (GMOs) as the Roundup Ready (RR) soya and Bt corn, the European food industry came face to face with the question of the use and labeling requirements on GMO crops and its derivatives. Although even today, no defined European legislation is available, a definitive need for detection methods exists. Both DNA and protein based methods have been developed and applied for the detection of RR soya beans and its derivatives. For the CP4 synthase, synthetic peptides corresponding with the antigenic and non-homologous parts of the CP4 synthase were synthesized and mono-specific anti-CP4 synthase monoclonal antibodies were prepared by hybridoma technology. The monoclonal antibodies were able to detect the CP4 synthase in the RR soya using Western blotting analysis. Detection limits were found between 0.5% and 1%. The method is currently validated for half-and final products. The applied DNA methodology was making use of polymerase chain reactions (PCR) using sets of primers along the gene encoding the Agrobacterium CP4 synthase. DNA extraction and purification conditions were examined on a case-by-case approach for a scala of soya products (lecithin, oil, soybean meal, soy protein isolates etc.), half-products and final consumer products. Detection limits were found between 0.01% and 0.1%. In this paper a comparison will be made between the two types of methods in relation to sample preparation, sensitivity, validation and the use for half-products and final consumer products.
A fast and quantitative method was developed to detect transgenic "Maximizer" maize "event 176" (Novartis) and "Roundup Ready" soybean (Monsanto) in food by real-time quantitative PCR. The use of the ABI Prism 7700 sequence detection system allowed the determination of the amplified product accumulation through a fluorogenic probe (TaqMan). Fluorescent dyes were chosen in such a way as to coamplify total and transgenic DNA in the same tube. Using real-time quantitative PCR, 2 pg of transgenic or total DNA per gram of starting sample was detected in 3 h after DNA extraction and the relative amounts of "Maximizer" maize and "Roundup Ready" soybean in some representative food products were quantified.
A new method for the specific, sensitive, and semiquantitative detection of pork (Sus scrofa) in heat-treated meat products has been developed. DNA was isolated from meat samples by using a DNA-binding resin and subjected to polymerase chain reaction (PCR) analysis. First, oligonucleotides yielding a specific 137-base-pair (bp) fragment from eucaryotic DNA amplified from a highly conserved region of the 18-S ribosomal gene was used to assess DNA quality. Second, the presence of pork DNA was determined with specific oligonucleotides yielding a 108-bp fragment amplified from the porcine growth hormone gene. The test detected pork in fresh or heated meat mixtures of pork in beef at levels below 2%. This approach was superior to commercially available immunological tests that were not able to detect levels of pork less than 20% in cooked meat or less than 10% in fresh meat.
A method has been developed to distinguish between traditional soy beans and transgenic Roundup Ready soy beans, i.e. the glyphosate ('Roundup') resistant soy bean variety developed by Monsanto Company. Glyphosate resistance results from the incorporation of an Agrobacterium-derived 5-enol-pyruvyl-shikimate-3-phosphatesynthase (EPSPS) gene. The detection method developed is based on a nested Polymerase Chain Reaction (PCR) procedure. Ten femtograms of soy bean DNA can be detected, while, starting from whole soy beans, Roundup Ready DNA can be detected at a level of 1 Roundup Ready soy bean in 5000 non-GM soy beans (0.02% Roundup Ready soy bean). The method has been applied to samples of soy bean, soy-meal pellets and soy bean flour, as well as a number of processed complex products such as infant formula based on soy, tofu, tempeh, soy-based desserts, bakery products and complex meat and meat-replacing products. The results obtained are discussed with respect to practical application of the detection method developed.
Biotechnology can contribute to future food security if it benefits sustainable small-farm agriculture in developing countries. Presently, agrobiotechnology research cites ethical, safety, and intellectual property rights issues. Protection of intellectual property rights encourages private sector investment in agrobiotechnology, but in developing countries the needs of smallholder farmers and environmental conservation are unlikely to attract private funds. Public investment will be needed, and new and imaginative public-private collaboration can make the gene revolution beneficial to developing countries. This is crucial for the well-being of today's hungry people and future generations.
http://www.jstor.org/stable/2898841
I n recent years, the U.S. agricultural community has adopted new plant varieties developed through modern technologies, including genetic transformation. Some of the new varieties include plants producing an insect-toxic protein from Bacillus thuringiensis and plants resistant to a popular
Nature Biotechnology journal featuring biotechnology articles and science research papers of commercial interest in pharmaceutical, medical, and environmental sciences.
Fumonisins B1 and B2 were determined in 42 samples of different maize products from the Swedish market by 2 different methods based on cleanup steps using an immunoaffinity column and a combination of SAX + C18 columns, respectively. A simple "precipitation step" was included before the samples were added to the main column(s), giving less column clogging, fewer interfering peaks, and better recoveries for the different sample matrixes. Recovery, repeatability, and results from the survey showed comparable results with the methods. The limit of detection for both methods was 5 micrograms/kg for fumonisin B1 and 10 micrograms/kg for fumonisin B2. All 7 maize chips analyzed and 6 of 8 popcorn samples contained fumonisins (B1 + B2) with averages of 180 and 115 micrograms/kg, respectively. All other samples except a maize flour sample contained little or no fumonisins.
In this study, the application of a qualitative and a quantitative method of analysis to detect genetically modified RR-Soy (Roundup-Ready Soy) in processed foods is described. A total of 179 various products containing soy such as baby food and diet products, soy drinks and desserts, tofu and tofu products, soy based meat substitutes, soy protein, breads, flour, granules, cereals, noodles, soy bean sprouts, fats and oils as well as condiments were investigated following the pattern of the section 35 LMBG-method L 23.01.22-1. The DNA was extracted from the samples and analysed using a soybean specific lectin gene PCR as well as a PCR, specific for the genetic modification. Additional, by means of PCR in combination with fluorescence-detection (TaqMan 5'-Nuclease Assay), suspicious samples were subjected to a real-time quantification of the percentage of genetically modified RR-Soy. The methods of analysis proved to be extremely sensitive and specific in regard to the food groups checked. The fats and oils, as well as the condiments were the exceptions in which amplifiable soy DNA could not be detected. The genetic modification of RR-Soy was detected in 34 samples. Eight of these samples contained more than 1% of RR-Soy. It is necessary to determine the percentage of transgenic soy in order to assess whether genetically modified ingredients were deliberately added, or whether they were caused by technically unavoidable contamination (for example during transportation and processing).
Validation of an immunoassay for the detection and quantification of Roundup-Ready® soybeans in food and food fractions by the use of reference material
- Lipp
Lipp, M. et al. (2000) Validation of an
immunoassay for the detection and
quantification of Roundup-Ready® soybeans in
food and food fractions by the use of reference
material. J. AOAC Int. 83, 99–127
Molecular Cloning: A Laboratory Manual Performance assessment under field conditions of a rapid immunological test for transgenic soybeans
- J Sambrook
- D J Russel
Sambrook, J. and Russel, D. (2000) Molecular
Cloning: A Laboratory Manual (3rd edn),
Cold Spring Harbor Laboratory Press
25 Fagan, J. et al. (2001) Performance assessment
under field conditions of a rapid immunological
test for transgenic soybeans. Int. J. Food Sci.
Technol. 36, 1–11
GMO Chip: Test Kit for the Detection of GMOs in Food Products, Cat. No. 5321300105 Quantitation of genetically modified organisms in food
GeneScan Europe (2001) GMO Chip: Test Kit for
the Detection of GMOs in Food Products,
Cat. No. 5321300105, Bremen, Germany
41 Hübner, P. et al. (1999) Quantitation of genetically
modified organisms in food. Nat. Biotechnol.
17, 1137–1138
Detection of genetically modified organisms with the polymerase chain reaction: potential problems with food matrices
- A Gasch
Gasch, A. et al. (1997) Detection of genetically
modified organisms with the polymerase chain
reaction: potential problems with food matrices.
In Foods Produced by Modern Genetic Engieering
(Schreiber, F.A. and Bögl, K.W., eds) 2nd Status
Report, pp. 90-79, BgVV-hefte