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Detection and traceability of genetically modified organisms in the food production
Abstract
Both labelling and traceability of genetically modified organisms are current issues that are considered in trade and regulation. Currently, labelling of genetically modified foods containing detectable transgenic material is required by EU legislation. A proposed package of legislation would extend this labelling to foods without any traces of transgenics. These new legislations would also impose labelling and a traceability system based on documentation throughout the food and feed manufacture system. The regulatory issues of risk analysis and labelling are currently harmonised by Codex Alimentarius. The implementation and maintenance of the regulations necessitates sampling protocols and analytical methodologies that allow for accurate determination of the content of genetically modified organisms within a food and feed sample. Current methodologies for the analysis of genetically modified organisms are focused on either one of two targets, the transgenic DNA inserted- or the novel protein(s) expressed- in a genetically modified product. For most DNA-based detection methods, the polymerase chain reaction is employed. Items that need consideration in the use of DNA-based detection methods include the specificity, sensitivity, matrix effects, internal reference DNA, availability of external reference materials, hemizygosity versus homozygosity, extrachromosomal DNA, and international harmonisation. For most protein-based methods, enzyme-linked immunosorbent assays with antibodies binding the novel protein are employed. Consideration should be given to the selection of the antigen bound by the antibody, accuracy, validation, and matrix effects. Currently, validation of detection methods for analysis of genetically modified organisms is taking place. In addition, new methodologies are developed, including the use of microarrays, mass spectrometry, and surface plasmon resonance. Challenges for GMO detection include the detection of transgenic material in materials with varying chromosome numbers. The existing and proposed regulatory EU requirements for traceability of genetically modified products fit within a broader tendency towards traceability of foods in general and, commercially, towards products that can be distinguished from each other. Traceability systems document the history of a product and may serve the purpose of both marketing and health protection. In this framework, segregation and identity preservation systems allow for the separation of genetically modified and non-modified products from "farm to fork". Implementation of these systems comes with specific technical requirements for each particular step of the food processing chain. In addition, the feasibility of traceability systems depends on a number of factors, including unique identifiers for each genetically modified product, detection methods, permissible levels of contamination, and financial costs. In conclusion, progress has been achieved in the field of sampling, detection, and traceability of genetically modified products, while some issues remain to be solved. For success, much will depend on the threshold level for adventitious contamination set by legislation.
... In this respect, nuclear technology is one of the most promising systems in food control, which is gaining more and more importance (Chen et al., 2017). 9.3 Traceability analysis 9.3.1 Immunoassays Immunochemical methods provide visual or instrumental measurement of the interaction of antibodies with protein molecules (Miraglia et al., 2004). Immunoassays can be considered as sensitive, dynamic, specific, and low-cost analytical methods for protein detection in complex structures (Brett, Chambers, Huang, & Morgan, 1999). ...
... The minimum threshold limit is only considered after the positive results of the first screening (Anklam, Gadani, Heinze, Pijnenburg, & Van Den Eede, 2002). Modern technologies focus on either the transgenetic DNA inserted or the novel proteins expressed in GMOs (Miraglia et al., 2004). ...
... Traceability of GMO can be achieved via protein-based or DNA-based analytical methods. The difference between protein-based and DNA-based analyses is the ability to detect a genetic modification by DNA-based analysis even if the modified gene is inactive in the cells (Miraglia et al., 2004). In general, there are two approaches as quantitative and threshold to detect GMO in foods (Stave, 1999). ...
Over the last decades, food safety has been a growing concern all over the world. Traceability systems are considered as the main tools for eliminating the concerns of consumers, manufacturers and legal authorities on food safety. Considering that, different types of techniques have been developed for the food industry to enable proper traceability. In this chapter, traceability techniques including document-based systems, information and communication technologies, alphanumerical codes, barcodes, holograms, radio-frequency identification (RFID), nuclear techniques, and nanotechnology are discussed in details. In addition, the major analysis methods that are used in food traceability, which include immunoassays, DNA-PCR methods, omics and isotope ratio analysis, are highlighted.
... The FAO and the WHO seem to be in the same picture. For the FAO/WHO (2001) cited in Miraglia et al. (2004) note that Codex Committee on Food Labelling is currently developing guidelines for labelling of GM foods. Due to the dangers and sensibilities of GMOs, the two organisations seek to trace GMOs (FAO/ WHO, 2003b cited in Miraglia et al. (2004). ...
... For the FAO/WHO (2001) cited in Miraglia et al. (2004) note that Codex Committee on Food Labelling is currently developing guidelines for labelling of GM foods. Due to the dangers and sensibilities of GMOs, the two organisations seek to trace GMOs (FAO/ WHO, 2003b cited in Miraglia et al. (2004). ...
... The FAO and the WHO seem to be in the same picture. For the FAO/WHO (2001) cited in Miraglia et al. (2004) notes that Codex Committee on Food Labelling is currently developing guidelines for labelling of GM foods (p. 1160). ...
... 1160). Due to the dangers and sensibilities of GMOs, the two organisations seek to trace GMOs (FAO/ WHO, 2003b cited in Miraglia et al. (2004). ...
This book aims to shed light on land grabbing, which corrupt politicians disguisedly call land leasing that many African countries are now involved in under the guise of investment. The major argument I make in this volume is that rich countries and companies are now committing a sacrilegious crime against poor Africans, which is likely to backfire soon than later after its adverse ramifications surface. To put it in context, such a crime is not only detrimental but self-inflicted wounds if not a suicidal act for Africa whose greedy and myopic rulers give a good name of investment. This sort of investment is not a panacea for Africa’s problem. Instead, it is a catalyst of land colonisation, which soon will exacerbate the problem. There are ways of solving Africa’s economic tanking.
The book poses a few simple questions: Instead of leasing out the land, why can’t such countries enter agreements with those they are leasing land to and produce food or whatever produce and supply them? Does Africa have any plan B in case things go skewwhiff in this agricultural geopolitics and land grabbing? Where did Africa study the entire project to assess its performance before swallowing it hook, line, and sinker for its peril. Also, the book considers landlessness in many African countries as a ticking bomb, especially when victims evidence how the land they call theirs is decadently and recklessly dished out by corrupt and irresponsible rulers or official in their countries while they are not only landless but also starving simply because those they wrongly trust have openly betrayed and sold them. Africa has always suffered from food insecurity. Land grabbing soon will become a food and national security issue.
... Traceability is a management system designed to maintain credible custody of animals or animal products by tracing their processes through various steps within the food chain, from production to sale [1,2]. Recently, traceability has become an accepted standard procedure in the agri-food industry. ...
The Nagoya breed, so-called Nagoya Cochin, is a native Japanese chicken. As the mislabeling of chickens is a matter of concern, causing economic losses relating to the production of the Nagoya breed, a simple and high-accuracy assay is required to distinguish the Nagoya breed. This study successfully developed a novel screening assay to discriminate the Nagoya breed using a high-resolution melting (HRM) assay. A primer set for HRM analysis was designed to amplify the CA repetition in the ABR0417 microsatellite marker. By comparing the results of the HRM analysis with those of the DNA sequence analysis, we investigated the applicability of the HRM-based assay using four differential chickens of the Nagoya breed and twelve other breeds. In the ABR0417 marker, the sequences of all four Nagoya breed chickens were identical to the ABR0417 reference sequence of the Nagoya breed, whereas the sequences for the twelve other chickens were different from those of the Nagoya breed chickens. The HRM analysis of the 16 chickens revealed that the melting curve and peak plots of the Nagoya breed chickens were distinct from those of other chickens. These results demonstrate that this HRM-based method is a simple genetic test to identify the Nagoya breed using the ABR0417 marker.
... The strategies may include simple testing for the presence of transgenic elements or GM events, or may be more complex matrix approaches aiming to assess the need, for instance, compliance with a labeling requirement or specifically targeting UGM. 16,17 Most of the GM testing laboratories commonly employ GMO screening approaches targeting common transgenic elements such as promoters for example, Cauliflower Mosaic Virus 35S promoter (P-35S), terminators (nopaline synthase terminator (T-nos), transgenes (cp4-epsps, cry genes, pat, bar) and so forth. in order to cover as much as possible GM events presence of which can be further confirmed at the subsequent step. Though these matrix approaches work efficiently targeting known/authorized events, they provide certain challenges in terms of detection and identification of UGM, which have been summarized as follows: ...
The approval status of a genetically modified (GM) event varies from country to country. GM events approved in one country (considered as authorized GM or AGM) may not necessarily have the same approval status in other countries (considered as unauthorized GM or UGM). Detecting UGM in the supply chain is a challenge as the genetic information is not always available. In India, four Bt cotton events are approved, whereas several other GM events have been imported for research purposes. Many food derivatives (non‐GM) are being imported from the countries where GM events of food crops are approved so it is necessary to track the unauthorized entry of GM products. Selected consignments or food products need to be checked for GM status for regulatory compliance. In farmers' fields, the chances of unintentional introgression or adventitious presence of transgenes also need to be monitored in a systematic manner. An appropriate strategy needs to be developed to check for UGM in the food and agricultural supply chain. In this article, approaches for UGM detection have been discussed with a focus on application in the Indian context. Detection methods based on the GMO matrix, multiplex PCR, real‐time PCR (qPCR), and loop‐mediated isothermal amplification (LAMP) could be employed keeping in view the regulatory requirement or practical application. For checking UGM with unknown genetic construct, methods such as next‐generation sequencing (NGS) may be employed. The advantages and disadvantages of the different approaches are discussed in the function of the analytical strategy and its application for control purposes.
... USA is one of the top producers of GMO, however, GM labeling in this country is still not mandatory, whereas it is mandatory in many Asian and European countries. 5 The Arabian countries' imports of food products are increasing; however, it is difficult to prohibit and control the imported food containing GM ingredients. On the other hand, consumption of GM food or food containing GM additives is still questionable from a public health point of view, especially in countries that depend upon the importation of food products where its control systems are not well developed to control the biosecurity of these food product imports. ...
Vegetable proteins such as soybean protein have numerous nutritional and functional characteristics, and consequently, their utilization in meat products development has dramatically increased in recent decades. Due to high demands for soybean, transgenic Roundup Ready (RR) soybean line grains were developed and widely distributed into global markets. The current study was designed to investigate the presence of transgenic soybean in meat products sold in Riyadh food retails, Saudi Arabia. After extraction of DNA from meat product samples, qualitative duplex polymerase chain reaction (PCR) was used to detect the genetically modified (GM) soybean products in the meat samples using pairs of primers targeting the lectin gene and the 35S promoter. Real-time PCR was used to quantify the percentage of RR soy products in the positive samples. The results clarified that out of 96 tested meat product samples (minced, burger, luncheon, canned, and sausages), 75 samples were positive for the presence of lectin gene, of which 42 samples representing 43.75% of total meat product samples were positive for the presence of 35S promoter. All positive samples for 35S promoter contained RR soy below 0.1%. The results of the consumer acceptance questionnaire of GM additives in meat products proved the presence of several critical aspects of concerns to consumers of meat products in different localities of Riyadh city.
... Molecular characterization of GMOs at the genome level often generates information on exogenous DNA integration and its inheritable stability, and a full understanding of exogenous DNA integration is a fundamental step in molecular characterization 3 . Exogenous DNA integration in GMOs includes integrated exogenous genes, integration sites, flanking DNA sequences of all inserts, the copy number of all inserts, and the presence or absence of unexpected vector backbone sequences 4 . ...
Efficient, accurate molecular characterization of genetically modified (GM) organisms is challenging, especially for those transgenic events transferred with genes/elements of recipient species. Herein, we decipher the comprehensive molecular characterization of one novel GM rice event G281 which was transferred with native promoters and an RNA interference (RNAi) expression cassette using paired-end whole genome sequencing (PE-WGS) and modified TranSeq approach. Our results show that transgenes integrate at rice chromosome 3 locus 16,439,674 included a 36 bp deletion of rice genomic DNA, and the whole integration contains two copies of the complete transfer DNA (T-DNA) in a head-to-head arrangement. No unintended insertion or backbone sequence of the transformed plasmid is observed at the whole genome level. Molecular characterization of the G281 event will assist risk assessment and application for a commercial license. In addition, we speculate that our approach could be further used for identifying the transgene integration of cisgenesis/intragenesis crops since both ends of T-DNA in G281 rice were from native gene or elements which is similar with that of cisgenesis/intrasgenesis. Our results from the in silico mimicking cisgenesis event confirm that the mimic rice Gt1 gene insertion and its flanking sequences are successfully identified, demonstrating the applicability of PE-WGS for molecular characterization of cisgenesis/intragenesis crops.
... A genetically modified plant is a plant whose genetic formation has been altered by means of the recombinant DNA technology (18). The genetic modification usually involves inclusion of a piece of DNA (the insert) taken from other naturally occurring organisms, a synthetic combination of different smaller pieces of DNA into the genome of the organism to be modified through the procedure of genetic transformation (11). ...
Aim of this research was quantitative detection of Increase and decrease in copy number of CaMV-35S promoter and Nos terminator in genetic modified tomato by using Real-time PCR. Twenty four samples of genetic modified tomato seeds were isolated from 84 tomato samples collected from Iraqi markets during the period from December 2016 to January 2017. The experiences were conducted in the Institute of Genetic Engineering, University of Baghdad. Go Taq®qPCR master mix kit supplied by USA Promega Company, three specific primers to CaMV-35S promoter, T-Nos terminator and β-actin housekeeping gene supplied by Canadian Alpha Company were used. To quantitative detection of increase and decrease in copy number of GM tomato samples contain CaMV-35S promoter and T-Nos terminator, comparing with the β-actin (houskeeping gene) using Multiple of Median (MoM) equation. The results showed that the lowest recorded of Ct value was (27.88) for CaMV-35S promoter gave an increase in copy number (1.1766) above the normal limit, while highest recorded of Ct value was (32.67) gave an increase in copy number (1.0350) above the normal limit. The lowest recorded of Ct value was (27.35) for T-Nos terminator gave an increase in copy number (1.1600) above the normal limit, whereas highest recorded of Ct value was (32.82) gave a decrease in copy number (0.9920) under the normal limit.
... A genetically modified plant is a plant whose genetic formation has been modified by means of the recombinant DNA technology (1). The genetic modification commonly involves inclusion of a piece of DNA (the insert) taken from other naturally occurring organisms, through the execution of genetic transformation (2). ...
The present study was focused to detect leading elements that control gene expression in genetically modified tomato by using conventional PCR technique.These common elements in all GM. plants are CaMV-35S promoter isolated from cauliflower mosaic virus and T-Nos terminator from the Agrobacterium tumefaciens. Seventy eight tomato genotypes were collected from Iraqiinstitutions and markets. The experiment was conducted in the Institute of Genetic Engineering/University of Baghdad/ Iraq and Directorate of Seeds Testing and Certification/Ministry of Agriculture/ Iraq. The tomato DNA samples were extracted manually by C-hexadecyl-Trimethyl-Ammonium-Bromide (CTAB) method. When measuring the optical density (OD) of the tomato samples, most purity values were found to be between (1.7-1.9).Two specific primers of CaMV-35S promoter, Nos terminator supplied by Canadian Alpha DNA Company, AccuPower®PCR Pre mix PCR supplied by Korean Bioneer Company and positive control (plasmid) supplied by Dr. ShathaAyidYousif/ Directorate of Agricultural Research/ Ministry of Science and Technology/ Iraq, were used in this study. Results showed that twenty four tomato genotypes were genetically modified. The primer specific of CaMV-35S promoter recorded a PCR product of 195 bp in 15 GM tomato and 13 GM tomato genotypes contain Nos terminator were a PCR product of 180 bp which as match with results of positive control (plasmid) which contains promoter and terminatorand that four tomato genotypes contain major components CaMV-35S promoter and Nos terminator together in the same sample.
... Currently, the most commonly used DNA-based detection techniques are PCR and qPCR. qPCR, the most commonly used DNA quantification method, has the advantages of low pollution and high specificity 28 . As shown in Figs. 6 and 7, clear bands in the mouse stomach were observed pre-gavage (0 min) and post-gavage (i.e., during 40-120 min). ...
This study aimed to perform qualitative and quantitative examination of DNA degradation during the digestion process in the mouse gut through PCR, qPCR and short tandem repeat (STR) analysis. Human blood leukocytes were gavaged into the digestive tract in mice. GAPDH, TH01, TPOX and D7S820 genes in the contents of the stomach and small intestine were analyzed with PCR and qPCR at various times pre- and post-gavage. Through STR analysis, 21 human genomic DNA loci were analyzed. The half-life of DNA degradation, and the relationship between the average peak area and digestion time were determined. The PCR results showed bands of amplified genes at pre-gavage (0 min) and post-gavage (40, 80 and 120 min) from the mouse stomach contents, whereas no DNA bands from small intestinal chyme were observed after gavage. The qPCR results revealed a significant decrease in DNA concentrations during 40–120 min in the mouse stomach after gavage. At 120 min, 85.62 ± 8.10% of the DNA was degraded, and the half-life of exogenous DNA degradation in the mouse stomach was 70.50 ± 5.46 min. At various digestion times, almost no target genes were detected in the mouse small intestinal chyme. STR analysis showed a decrease in allele numbers with bowel advancement in the small intestine in mice. The degradation of exogenous DNA was higher in the mouse stomach during the first 2 h, and almost complete degradation was observed within 40 min after entering the small intestine in mice.
... Therefore, obtaining the data of robust molecular characterization on the insertion positions of the transgenes and their flanking sequences is valuable for the developers and regulators of GM crops and the risk assessment of the latter. Such data are also useful for the development and validation of the specific detection methods for the monitoring of commercial genetically modified organisms (GMOs) (Miraglia et al., 2004, andFraiture et al., 2015). ...
The insertion position of the exogenous fragment sequence in a genetically modified organism (GMO) is important for the safety assessment and labeling of GMOs. SK12-5 is a newly developed transgenic maize line transformed with two trait genes [i.e., G10evo-5-enolpyrul-shikimate-3-phosphate synthase (EPSPS) and Cry1Ab/Cry2Aj] that was recently approved for commercial use in China. In this study, we tried to determine the insertion position of the exogenous fragment for SK12-5. The transgene–host left border and right border integration junctions were obtained from SK12-5 genomic DNA by using the thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and next-generation Illumina sequencing technology. However, a Basic Local Alignment Search Tool (BLAST) analysis revealed that the flanking sequences in the maize genome are unspecific and that the insertion position is located in a repetitive sequence area in the maize genome. To locate the fine-scale insertion position in SK12-5, we combined the methods of genetic mapping and nanopore-based sequencing technology. From a classical bulked-segregant analysis (BSA), the insertion position in SK12-5 was mapped onto Bin9.03 of chromosome 9 between the simple sequence repeat (SSR) markers umc2337 and umc1743 (26,822,048–100,724,531 bp). The nanopore sequencing results uncovered 10 reads for which one end was mapped onto the vector and the other end was mapped onto the maize genome. These observations indicated that the exogenous T-DNA fragments were putatively integrated at the position from 82,329,568 to 82,379,296 bp of chromosome 9 in the transgenic maize SK12-5. This study is helpful for the safety assessment of the novel transgenic maize SK12-5 and shows that the combined method of genetic mapping and the nanopore-based sequencing technology will be a useful approach for identifying the insertion positions of transgenic sequences in other GM plants with relatively large and complex genomes.
... This study adopts a choice experiment model on the GM soybean oil sales and labelling in China Including Regulations on Administration of Agricultural Genetically Modified Organisms Safety and Administrative Measures for Agricultural GMOs Labeling issued by the Ministry of Agriculture and Rural Affairs of P. R. China. This study follows the relevant literature [26,27,35,[37][38][39][40], and the representative GM organism safety management policies. The CE model contained three labelling attributes and the payment vehicle (Table 1), that is, allergen presence labelling, nutrient and compositional change labelling, traceability codes, and price. ...
This study investigates consumers’ preferences for mandatory labelling conveying the health and safety attributes of genetically modified soybean oil. The enhanced mandatory labelling includes allergen presence labelling, nutrient and compositional change labelling and traceability codes. The data were collected from a consumer survey in the eastern, central and western regions of China, with a total sample size of 804 respondents. We evaluated consumer willingness to pay (WTP) for enhanced mandatory labelling using a choice experiment approach. The results show that Chinese consumers are most favorable to traceability codes with a WTP of RMB 8.92, followed by allergen presences labelling, with RMB 6.57. Eastern consumers would like to pay a higher premium for the three types of enhanced mandatory labelling information, while central consumers only show a positive preference for traceability codes. The results imply that the efforts and policy strategies for enhanced mandatory labelling will benefit residents. Further studies can be expended to other genetically modified (GM) foods. This study provides information for the agency to improve mandatory GM food labelling management. This paper contributes to the growing body of the GM food literature by explicitly investigating consumer preference and WTP for mandatory labelling conveying the health and safety attributes of the GM foods.
... The event-specific screening method uses the unique sequences at the junction of 5′ or 3′ flanking region of genomic DNA and inserted gene. Therefore, the above-mentioned method is very specific and has become the most important method for the identification of GM crops (11,12). However, the information regarding the junction areas in different GM crops is not always available. ...
Among the genetically modified (GM) crops that are being approved for commercialization, herbicide resistant crops, especially those harboring cp4-epsps, have a considerable contribution. Gene-specific methods can be used to screen the presence of GMOs. To establish an effective qualitative and quantitative screening method, a set of primers were designed considering the cp4-epsps sequence. The specificity, the limit of detection, the efficiency, and the ability to quantify the GMO content were tested in GM cotton, soybean, and canola events. The results demonstrated that the primers can specifically detect cp4-epsps GM crops. The limit of detection was found to be 0.4 ng /μl DNA per PCR reaction with the ability to detect 1-16 copies of the haploid genome of each GM event. The efficiency of this screening method (which was 94-110 % with an R2 higher than 0.96) indicated that these new primers can be applied to the screening of GM samples that contain the cp4-epsps gene. Also, the gene-specific real-time PCR screening method could be successfully developed for qualification of different types of GM cotton, soybean and canola events with the construction of a serial dilution ranging from 10 % to 1 %.
... [a] Based on the GM food sales and labeling requirements in China, the traceability is selected as one type of enhanced mandatory labeling. Following relevant literature 13,17,[29][30][31][32][33] and the representative GMO safety management policies, the traceability system has been established to document the entire process of GM soybean oil production in China. The system facilitates easy separation of the GM soybean oil from their non-GM counterparts "from farm to fork," and well serves the purpose of marketing and health protection. ...
Consumer preference for the mandatory labeling of genetically modified (GM) foods promotes public support for the implementation of GM food policies. This study analyzes consumers’ preference for the traceability of GM soybean oil. Survey data were collected through a self-administered survey covering 804 randomly sampled urban residents in the eastern, central and western regions of China. Using a logit model, this analysis examines the impacts of influential factors on consumers’ preference for traceability. The results show that about 56.5% of the respondents have a positive preference for the traceability of GM soybean oil. Factors increasing the preference for traceability include a better perception of the attributes of nutrition benefit and potential health risk, perceived inadequacy of simple mandatory labels, more attention paid to food labels, and distrust in the agencies overseeing GM food safety. Enhancing consumers’ perceptions of GM-related attributes and awareness of food labels will help improve the mandatory labeling management of GM foods.
... Different processing conditions, for example, high temperature, extrusion or pH can lead to DNA degradation (Gryson, 2010;Bauer et al., 2004). However, presence of GMO can still be detected by polymerase chain reaction (PCR) for DNA fragments shorter than 200 bp (Bergerova et al., 2010;Miraglia et al., 2004). PCR as a J o u r n a l P r e -p r o o f DNA-based method is the most critical means for GMO analysis in food and feed, including conventional PCR, real-time PCR, multiplex PCR and digital PCR (Meyer, 1999;Costa et al., 2010;Marmiroli et al., 2008;Park et al., 2017;Zhang et al., 2018;Bogožalec et al., 2019). ...
To develop a simple and fast method for screening genetically modified ingredients from processing by-product and waste, direct quantitative PCR (qPCR) kit-Taqman which omitting multi genomic DNA preparing steps was developed in this study. A total of 18 oil crop processing by-products and wastes including 10 soybean and 8 cotton materials were collected from food processing factories. Compared with 2 commercial direct qPCR kits, conditions of DNA releasing procedure and PCR amplification were optimized. Element screening was performed at the initial step of genetically modified (GM) ingredient testing procedure via direct qPCR. GM event identification was carried out in positive samples by initial screening. In initial screening, 5 screening elements (P-35S, T-NOS, Cp4-epsps, bar and pat) for soybean materials and 6 screening elements (P-35S, T-NOS, NPTII, Cry1Ac, bar and pat) for cotton samples were detected. In GM event identification, MON531 and MON1445 were found in cotton materials. Results were further confirmed by real-time PCR with DNA extraction and purification. The direct qPCR system proposed by this research was convenient for rapid screening and identification of GM ingredients in oil crop primary by-product and waste.
... PCR-based detection can be evaluated in at least four categories related to the level of specificity namely screen-specific, genespecific, construct-specific, and event-specific evaluation. The first category include common DNA elements in GMOs, such as promoters and terminators that are present in many different GMOs (Holst-Jensen, 2009, Miraglia et al., 2004. Sometimes, marker genes are also used as screening targets, such as gene encoding resistance to ampicillin (bla) and neomycin/kanamycin (nptII) antibiotics used in selection. ...
Despite the controversy over GM food and feed, these products are still on
the market. Iraqi Biosafety Law had not yet come into force and no law has so far
been enacted on the labeling of genetically modified food and feed. To our
knowledge there is no available quantitative data on the prevalence of GM crops in
feeds in Iraq. The aim of the present study was to detect the presence of GMO
feeds by PCR based method. Five fodder kinds from various markets in Iraq were
chosen as materials. Feed1 (seed mixture; including safflower, flax, millet with
colorful protein supplements), feed 2 which feed crushed, feed 3 (seeds mixture;
including wheat, corn, sorghum and sunflower), feed 4 which is grain mixture with
main barley seeds and feed 5 which is a fodder pellet. The screening of all samples
was performed using the primers for Chloroplast rbcL (internal control),
CaMV35S promoter, NOS terminator, Cry1Ac and EPSPS genes. The study
proved the existence of genetic modification in all samples. For confirmation
procedures the amplified fragments corresponding to the EPSPS were verified by
sequencing.
... 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. Zein is used for the detection of genetic modification in maize. ...
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.
... This technique allows for the estimation of both quantity and size of the DNA. Restriction digestion of the DNA or DNA sequencing may also be used to further evaluate the accurate identity of the amplified DNA (Miraglia et al., 2004). The choice of the desired DNA regions to be amplified relies on the specificity level needed for GMO identification (Randhawa et al., 2016). ...
Genetically modified organisms (GMOs) are receiving attention worldwide mainly due to concerns regarding their safety. Various countries have set certain regulations that GMOs must be labeled prior to their release. For proper monitoring of GMOs, the presence and the amount of GM material must be verified via the use of efficient and accurate detection methods. This chapter describes the different novel analytical methods which allow for high throughput diagnosis of GMOs. Among these detection methods, capillary-gel-electrophoresis, digital-PCR, microarray and next-generation sequencing is being well adopted for their accuracy and precise detection of GM-event.
... The challenge for these growers is to keep AP levels within the threshold, which involves steps like minimizing cross-fertilization between neighboring farms (Demont and Devos, 2008). Several European studies have identified and tested management strategies like temporal and spatial strategies to cut down the adventitious presence to a minimum (Miraglia et al., 2004;Coleno et al., 2009). Use of certified seeds, spatially isolating farms, implementing pollen barriers, scheduling different timing for sowing and flowering periods, cleaning agricultural and material handling machinery, and clustering of fields are some measures that could be taken at the production facilities to lessen the contamination of GM crops in non-GM crops (Devos et al., 2009;Mosher and Hurburgh, 2010). ...
Highlights Keywords: Corn, Genetic modification, Graphical User Interface (GUI), Threshold level.
A GUI tool was developed to predict the adventitious presence in non-GM produce. Keywords: Corn, Genetic modification, Graphical User Interface (GUI), Threshold level.
The software calculates tolerance and the probability of GM corn in non-GM corn. Keywords: Corn, Genetic modification, Graphical User Interface (GUI), Threshold level.
Predicted probability of contamination ranged from 0.050 to 0.356 at tolerance levels ranging from 0.1% to 5.0%. Keywords: Corn, Genetic modification, Graphical User Interface (GUI), Threshold level.
Keywords: Corn, Genetic modification, Graphical User Interface (GUI), Threshold level.
Abstract. The current rate of population growth necessitates the use of viable technologies like genetic modification to address estimated global food and feed requirements. However, in recent years, there has been an increase in resistance against the diffusion of genetic modification technology around the world. Many countries have adopted coexistence policies to allow a certain percentage of adventitious presence in non-genetically modified crops. However, the tolerance percentage for adventitious presence has been a bottleneck to free trade in some cases. It is a challenging task to fix a tolerance percentage considering the level of permeation of genetic modification technology in agriculture. This article introduces a software developed to serve as a decision-making tool to predict the probability distribution of genetically modified (GM) contamination in non-GM grain lot using user inputs such as final quantity of processed corn, overall tolerance level, and moisture content. The output from the software includes the mass of corn in each processing stage, the tolerance level and the probability distribution of potential GM contamination. The software predicted the probability of contamination with adventitious presence at tolerance levels of 5.0%, 3.0%, 1.0%, 0.9%, 0.5%, and 0.1% as 0.05, 0.07, 0.11, 0.12, 0.16, and 0.36, respectively. The predictions from the model were compared to a similar study wherein the effect of tolerance levels incurred in the costs of segregation was studied. The mean absolute percentage error for the predictions was found to be 3.07%. This software can be used as a tool in testing GM contamination in non-GM grain against a desired threshold levels in a grain elevator. Keywords: Corn, Genetic modification, Graphical User Interface (GUI), Threshold level.
... Since the introduction of the labelling rules of GM products, different analytical methodologies have been used to identify the GMOs. The most accurate and commonly used detection methods implemented in different laboratories are based on the amplification of the specific DNA targets by using the PCR technique (MIRAGLIA, 2004;RODRÍGUEZ-LÁZARO, 2007). Cauliflower Mosaic Virus 35S promoter (CaMV P-35S) and Nopaline Synthase Terminator (T-NOS) are the most widely used transgenic elements in the usual GM events (RUTTINK et al., 2010). ...
With the ever-increasing number of GM crops introduced to the market, it is
required to respect customers’ freedom of choice and their perception toward
GMOs. According to obligatory demand by food and drug administration for GM
testing on raw and processed materials, efficient and robust analytical
approaches are needed for screening and identification of GM crops. This
article describes the development and applicability of a PCR-based GMO
testing of the most frequent constructs used in transgenic soy, corn and
rice varieties which include CaMV35S promoter and the NOS terminator and
also plant endogenous sequences Lectin, Zein, SPS as internal controls. A
total of 2866 different sample inputs collected during the year 2015 to 2017
including GM and non-GM materials have been tested. Results obtained from
qualitative PCR test on rice, maize and soybean-containing food samples
showed that in total, 1.59% of maize samples and 7.53% of soya samples were
GM positive and no GM positive rice sample was detected. The presented
method showed high specificity and sensitivity offering an absolute limit of
detection of 0.25% GM construct in the samples and sufficient
reproducibility of the method was proved. This method fits the purpose of
GMO testing laboratories due to its convenience and robustness.
... A genetically modified plant is a plant whose genetic formation has been altered by means of the recombinant DNA technology (18). The genetic modification usually involves inclusion of a piece of DNA (the insert) taken from other naturally occurring organisms, a synthetic combination of different smaller pieces of DNA into the genome of the organism to be modified through the procedure of genetic transformation (11). ...
Aim of this research was quantitative detection of Increase and decrease in copy number of CaMV-35S promoter and Nos terminator in genetic modified tomato by using Real-time PCR. Twenty four samples of genetic modified tomato seeds were isolated from 84 tomato samples collected from Iraqi markets during the period from December 2016 to January 2017. The experiences were conducted in the Institute of Genetic Engineering, University of Baghdad. Go Taq®qPCR master mix kit supplied by USA Promega Company, three specific primers to CaMV-35S promoter, T-Nos terminator and β-actin housekeeping gene supplied by Canadian Alpha Company were used. To quantitative detection of increase and decrease in copy number of GM tomato samples contain CaMV-35S promoter and T-Nos terminator, comparing with the β-actin (houskeeping gene) using Multiple of Median (MoM) equation. The results showed that the lowest recorded of Ct value was (27.88) for CaMV-35S promoter gave an increase in copy number (1.1766) above the normal limit, while highest recorded of Ct value was (32.67) gave an increase in copy number (1.0350) above the normal limit. The lowest recorded of Ct value was (27.35) for T-Nos terminator gave an increase in copy number (1.1600) above the normal limit, whereas highest recorded of Ct value was (32.82) gave a decrease in copy number (0.9920) under the normal limit.
... Those result indicated that 26 and 44% of samples containing soybean and maize were positive respectively, in contrast, all of the rice samples were negative for these two primers [9]. Other researchers also concluded that successful screening of GMO targets in food products by PCR is achievable and preferred than other methods [32,33]. In our study, 2 out of 81 rice samples showed positive results for primer pair P35S-cf3/P35S-cr4, therefore it may to be genetically modified, while none of them has been labeled. ...
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.
... The onset of an intensification of research on genetics happened after the detection of a BT toxin-resistant caterpillar on a plantation, which means that in a short time, the insects and pests adapted to the genetically modified toxin produced by the plants. Miraglia et al. (2004) proposed adaptations in European legislation, with requirements in the detection and traceability of GMOs in food production chains. ...
Purpose
The purpose of this paper is to identify the relationship between the frequency of publication on food supply chain (FSC) traceability and the occurrence of foodborne diseases outbreaks.
Design/methodology/approach
A systematic review of the literature was carried out to locate the main articles published in the literature, followed by a content analysis in order to list the main food traceability technologies and their evolutions. Finally, a Spearman’s ρ correlation analysis between the frequency of publications on FSC traceability and the annual occurrence of foodborne outbreaks in the five largest food exporting countries in the world was performed.
Findings
In these analyses, the tools of radiofrequency, deoxyribonucleic acid, wireless sensor network, hazard analysis and critical control points and Internet of Things are the most researched technologies, and they are relevant in the evolution of traceability in the FSC. With correlation coefficients above 0.700 at 0.01 significance levels, this evolution of food traceability technologies has been one of the factors reducing the number of food outbreaks in the USA and Germany, countries with greater development of the health system and food control.
Originality/value
This paper provides an evaluation of the food traceability technologies and the effects of their evolutions in the occurrence of food outbreaks. This may help in the proposal of public policies related to food and outbreak control.
... A genetically modified plant is a plant whose genetic formation has been modified by means of the recombinant DNA technology (1). The genetic modification commonly involves inclusion of a piece of DNA (the insert) taken from other naturally occurring organisms, through the execution of genetic transformation (2). ...
The present study was focused to detect leading elements that control gene expression in genetically modified tomato by using conventional PCR technique.These common elements in all GM. plants are CaMV-35S promoter isolated from cauliflower mosaic virus and T-Nos terminator from the Agrobacterium tumefaciens. Seventy eight tomato genotypes were collected from Iraqi institutions and markets. The tomato DNA samples were extracted manually by C- hexadecyl- Trimethyl-Ammonium-Bromide (CTAB) method.. Results showed that twenty four tomato genotypes were genetically modified.The primer specific of CaMV-35S promoter recorded a PCR product of 195 bp in 15 GM tomato and 13 GM tomato genotypes contain Nos terminator were a PCR product of 180 bp which as match with results of positive control (plasmid) which contains promoter and terminator and that four tomato genotypes contain major components CaMV-35S promoter and Nos terminator together in the same sample.
Key words:GM Tomato, Conventional PCR, CaMV-35S promoter and T-Nos terminator
... Those result indicated that 26 and 44% of samples containing soybean and maize were positive respectively, in contrast, all of the rice samples were negative for these two primers [9]. Other researchers also concluded that successful screening of GMO targets in food products by PCR is achievable and preferred than other methods [32,33]. In our study, 2 out of 81 rice samples showed positive results for primer pair P35S-cf3/P35S-cr4, therefore it may to be genetically modified, while none of them has been labeled. ...
Background:
Composting plant leachate is considered as one of the highly polluted wastewaters which is necessary to be treated by simple, economic, fast and environmentally compatible methods. In this study, treatment of fresh composting plant leachate by electro-Fenton (EF) process was investigated.
Methods:
The effect of various input variables like pH (2-7), DC currents (1.5-3 A), H2O2 concentrations (theoretical ratio H2O2/COD: 0.1-0.6), TDS changes (4-6%), feeding mode, and BOD/COD ratio at the optimal point were studied. The settling characteristics of the waste sludge produced by the treatment (sludge volumes after 30-min sedimentation: V30) were also determined. Artificial neural network (ANN) approach was used for modeling the experimental data.
Results:
Based on the results, the best removal rate of COD was obtained at pH: 3, 3 A constant DC current value, 0.6 theoretical ratio H2O2/COD and the feeding mode at four step injection. BOD/COD ratio at the optimal point was 0.535 and the maximum COD removal was achieved at TDS = 4%. In the optimal conditions, 85% of COD was removed and BOD/COD ratio was increased from 0.270 to 0.535. The data follow the second-order kinetic (R2 > 0.9) and neural network modeling also provided the accurate prediction for testing data.
Conclusion:
Results showed that EF process can be used efficiently for treatment of composting plant leachate using the proper operating conditions.
... The increase in GM crop production has been coupled with an intricate and asynchronous international regulatory approval system, requiring identification and testing of food and agricultural products for the presence of GM content to simplify international trade. Molecular identification of GM crops confirms the identity and type of modified product at each stage and assures compliance with import for GM food and feed [14]. The testing of GM crops could be performed in open field or under controlled laboratory conditions that depends upon type of samples and sensitivity of test performed. ...
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.
... A genetically modified plant is a plant whose genetic formation has been altered by means of the recombinant DNA technology (18). The genetic modification usually involves inclusion of a piece of DNA (the insert) taken from other naturally occurring organisms, a synthetic combination of different smaller pieces of DNA into the genome of the organism to be modified through the procedure of genetic transformation (11). ...
The present study was focused to detect leading elements that control gene expression in genetically modified tomato by using conventional PCR technique.These common elements in all GM. plants are CaMV-35S promoter isolated from cauliflower mosaic virus and T-Nos terminator from the Agrobacterium tumefaciens. Seventy eight tomato genotypes were collected from Iraqi institutions and markets. The experiment was conducted in the Institute of Genetic Engineering/University of Baghdad/ Iraq and Directorate of Seeds Testing and Certification/Ministry of Agriculture/ Iraq. The tomato DNA samples were extracted manually by C- hexadecyl- Trimethyl-Ammonium-Bromide (CTAB) method. When measuring the optical density (OD) of the tomato samples, most purity values were found to be between (1.7-1.9).Two specific primers of CaMV-35S promoter, Nos terminator supplied by Canadian Alpha DNA Company, AccuPower®PCR Pre mix PCR supplied by Korean Bioneer Company and positive control (plasmid) supplied by Dr. ShathaAyidYousif/ Directorate of Agricultural Research/ Ministry of Science and Technology/ Iraq, were used in this study. Results showed that twenty four tomato genotypes were genetically modified.The primer specific of CaMV-35S promoter recorded a PCR product of 195 bp in 15 GM tomato and 13 GM tomato genotypes contain Nos terminator were a PCR product of 180 bp which as match with results of positive control (plasmid) which contains promoter and terminator and that four tomato genotypes contain major components CaMV-35S promoter and Nos terminator together in the same sample.
Key words:Tomato, Conventional PCR, CaMV-35S promoter and T-Nos terminator.
Introduction
Genetic engineering has revolutionized agriculture by transforming biotic and abiotic stress-resistance genes in plants. The biosafety of GM crops is a major concern for consumers and regulatory authorities.
Methodology
A 14-week biosafety and toxicity analysis of transgenic cotton, containing 5 transgenes ((Cry1Ac, Cry2A, CP4 EPSPS, VIP3Aa, and ASAL)), was conducted on albino mice. Thirty mice were divided into three groups (Conventional, Non-transgenic, without Bt, and transgenic, containing targeted crop) according to the feed given, with 10 mice in each group, with 5 male and 5 female mice in each group.
Results
During the study, no biologically significant changes were observed in the non-transgenic and transgenic groups compared to the control group in any of the study’s parameters i.e. increase in weight of mice, physiological, pathological, and molecular analysis, irrespective of the gender of the mice. However, a statistically significant change was observed in the hematological parameters of the male mice, while no such change was observed in the female study group mice. The expression analysis, however, of the TNF gene increases many folds in the transgenic group as compared to the non-transgenic and conventional groups.
Conclusion
Overall, no physiological, pathological, or molecular toxicity was observed in the mice fed with transgenic feed. Therefore, it can be speculated that the targeted transgenic crop is biologically safe. However, more study is required to confirm the biosafety of the product on the animal by expression profiling.
An integral component of the management system in the field of food safety is the examination of food products, which is based mostly on physical, chemical, physico-chemical and biochemical methods of research. Progress in the mastery of DNA diagnostic methods has become an incentive for the development and introduction into laboratory practice of highly sensitive methods for assessing the safety and quality of foodstuff, based on the polymerase chain reaction (PCR) method. In recent decades, the demand for molecular tools for food examination, authentication and traceability has increased significantly. This is due to the fact that legislation in the food sector is becoming increasingly strict, and market strategies are aimed at evaluating the food chain "from field to table" and ensuring that consumer choices match their expectations. An overview of proven and widely tested molecular approaches for the examination of food products is presented: PCR-RFLP method, RAPD-PCR, SSR-PCR, RTPCR. The potential and prospects of the latest technologies, such as SNP - single nucleotide polymorphisms, isothermal amplification, digital PCR, Whole-Genome Sequencing (WGS), DNA metabarcoding, are also described. The specified methods are characterized by high productivity, speed and scaling, enabling the study of biological systems at a new qualitative level. Examples of successful use of the specified methods for examination of foodstuff of plant and animal origin, their authentication and traceability are given. A broad panel of molecular methods is a powerful tool to protect both producers and consumers, providing consumers with freedom of choice and increasing transparency in food production systems, enabling honest producers to properly promote their products. Key words: DNA-technologies, polymerase chain reaction, food safety, foodstuff examination.
The cultured meat industry is continuously evolving due to the collective efforts of cultured meat companies and academics worldwide. Though still technologically limited, recent reports of regulatory approvals for cultured meat companies have initiated the standards-based approach towards cultured meat production. Incidents of deception in the meat industry call for fool-proof authentication methods to ensure consumer safety, product quality, and traceability. The cultured meat industry is not exempt from the threats of food fraud. Meat authentication techniques based on DNA, protein, and metabolite fingerprints of animal meat species needs to be evaluated for their applicability to cultured meat. Technique-based categorization of cultured meat products could ease the identification of appropriate authentication methods. The combination of methods with high sensitivity and specificity is key to increasing the accuracy and precision of meat authentication. The identification of markers (both physical and biochemical) to differentiate conventional meat from cultured meat needs to be established to ensure overall product traceability. The current review briefly discusses some areas in the cultured meat industry that are vulnerable to food fraud. Specifically, it targets the current meat and meat product authentication tests to emphasize the need for ensuring the traceability of cultured meat.
The main advantage of digital PCR (dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve. Crystal droplet dPCR has a three-color staining detection function, which enables multiplex PCR reaction. In this study, this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708×MON89788 with stacked traits. Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708×MON89788 soybean. Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.
Currently, science and technology are continuously evolving by convergence with each other. In agriculture, new concepts such as smart farm, vertical farming, and urban agriculture have emerged beyond the traditional form. Among the various types of smart farms, vertical farms are considered one of the most advanced forms of agriculture, and research and related industries are rapidly increasing. However, vertical farming has several limitations. The biggest problem is that the types of crops grown within this system are extremely limited. Industrially, only minimal crops are cultivated, and a significant number of many crops are rarely attempted due to cultural or economic limitations. This is especially the case with fruit crops because the innate form and various traits of fruit crops are not suitable for vertical farming. Therefore, this review will discuss the attributes of which fruit crops need to be improved to be grown on vertical farms. Finally, we show how the latest biotechnology and breeding techniques can enable the fast and accurate development of crops tailored to vertical farms.
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.
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.
Floriculture industry in India has been a sunrise industry and high growth industry with 100% export oriented status by the Government of India. It has been identified as one of the industry with an opportunity to grow and contribute positively to the economy of India and also had impressed major corporates in India. Floriculture industry should also adopt an effective traceability system which has been implemented by Agricultural and Processed Food Products Export Development Authority (APEDA) and its association with global standards (GS1), in the form of AnarNet, HortiNet, Peanut.Net, and GrapeNet for Indian farming products that are actively working in India. As there are both small and medium farmers involved in the floriculture industry there is inadequate market facility, high market fees, dishonesty of traders and Still India is in the early stage without proper supply chain, cold chain and transportation. So the main aim of this paper is to implement the multi-labeling traceability techniques in floriculture industry that helps the small, medium and large farmers to maintain quality and quantity, manage risks, proper flow of information and also helps in the development of floriculture supply chain management in India.
Arı Yetiştiriciliği Sorunları ve Çözüm Önerileri
The food supply chainFood supply chain (FSC) is complex, immense and crucial to human beings. Disruptions, fragmentation, poor product traceability, improper product flows, food contamination, food recall, etc., are some of the prevailing issues in the food supply chainFood supply chain. Many cases of E. Coli, hog contamination and halal meat contamination are also evident. This creates a strong need to identify, understand and resolve the issues, leading to an efficient and responsive food supply chain. This study identifies the various issues in the food supply chain and thereafter focuses on traceability and transportation-related issues. The authors have tried to conduct a review of the issues in the food supply chain, especially traceability, transportationTransportation and solutions under the purview of supply chain driversDrivers. The study can help future researchers identify and understand an issue and develop a feasible solution for it.
The genetically modified (GM) maize DBN9936 that has been granted a biosafety certificate needs to be regulated in China. An event-specific PCR assay targeting the junction region between the left border of its T-DNA and the flanking genomic DNA (gDNA) was established to be able to specifically identify the DBN9936 event from other GM maize and non-GM maize. Both qPCR and duplex ddPCR achieved accurate measurement of DBN9936 content in blind matrix and gDNA samples with acceptable precision in terms of a relative standard deviation (RSD) of <25% and a trueness in terms of relative bias of ±25%. The quantitative values of the same sample did not show significant difference between qPCR and duplex ddPCR, whereas the duplex ddPCR showed advantage over qPCR in terms of precision. This study provided an event-specific PCR method for identification and quantification of DBN9936 on both a real-time PCR platform and a ddPCR platform.
Efficient, accurate molecular characterization of genetically modified (GM) organisms is challenging, especially for novel transgenic products of cisgenesis/intragenesis transferred with genes/elements of recipient species. Herein, GM rice event G281, involving transfer with native promoters and an RNA interference (RNAi) expression cassette in a process similar to intragenesis , was subjected to molecular characterization using paired-end whole genome sequencing (PE-WGS). The results showed that transgenes integrated at rice chromosome 3 locus 16,439,674 included a 36 bp deletion of rice genomic DNA, and the whole integration contained two copies of the complete transfer DNA (T-DNA) in a head-to-head arrangement. No unintended insertion or backbone sequence of the transformed plasmid were observed at the whole genome level. Molecular characterization of the G281 event will assist risk assessment and application for a commercial license. Additionally, the findings demonstrate the applicability of PE-WGS for molecular characterization of cisgenesis / intragenesis crops.
Objective: Qualitative and quantitative examination of DNA degradation during the digestion process in the mouse gut through PCR, qPCR and short tandem repeat (STR) analysis.
Methods: Human blood leukocytes were gavage into the digestive tract of mice. GAPDH, TH01, TPOX and D7S820 genes in the contents of the stomach and small intestine were analyzed through PCR and qPCR at various time pre- and post-gavage. Through STR analysis, 21 human genomic DNA loci were analyzed. The half-life of DNA degradation, and the relationship between the average peak area and digestion time were determined.
Results: The PCR results showed DNA bands at pre-gavage (0 min) and post-gavage (40, 80 and 120 min) from the mouse stomach contents, whereas no DNA bands from small intestinal chyme were observed after gavage. The qPCR results revealed significant decrease in DNA concentrations during 40-120 minutes in mouse stomach after gavage. At 120 min, 85.62±8.10% of the DNA was degraded while the half-life of exogenous DNA degradation in mouse stomach was 70.50±5.46 min. At various time of digestion , almost no target genes were detected in the mouse small intestinal chyme. STR analysis showed a decrease in allele numbers with the advancement of bowl in the small intestine of mice.
Conclusions: The degradation of exogenous DNA was higher in mouse stomach during first two hours while almost complete degradation was noted in the small intestine of mice within 40 min.
The emerging use of genetic engineering technology led to the establishment of the Cartagena Protocol on Biosafety in 2001. India and Malaysia are signatories to the Protocol, having established regulatory measures governing the use of biotechnological genetic modification including regulation of genetically engineered crops from research to open cultivation and post-market surveillance. India and Malaysia have developed biosafety policies that display some similarities but also many differences, consequently impacting the practicalities of applying the technology to development and deployment of new crop varieties. The objective of this paper is to compare biosafety policies and regulatory frameworks that India and Malaysia have in place for the use of modern biotechnology. We highlight the implications of imposing rigid requirements as well as lacking harmonized policies on the approval process and trade flows, identifying these as potential barriers to the optimal use of modern crop biotechnology. We also briefly discuss how current interpretations of Living Modified Organisms and Genetically Modified Organisms in India and Malaysia will influence the pace of crops developed from new plant breeding techniques and propose options to regulate these technologies based on experience from other countries.
CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR associated system Cas9 (CRISPR/Cas9) system was discovered as the bacterial immune system against invading viruses. Its applications not only include genome editing but it is also being used for gene regulation, epigenome editing, chromatin engineering, and imaging. The most widely used CRISPR/Cas9 system which have been used for experimental genome editing consists of two components, i.e. Cas9 nuclease protein and sgRNA (a 20 nucleotide (nt) long RNA molecule). The programmability and specificity of this system is conferred by the gRNA molecule. These two components can either be delivered in the form of ribonucleoprotein complex (RNPs), in vitro transcribed mRNA or in the form of DNA which is transcribed in vivo to produce Cas9 and sgRNA. The efficiency of this system depends upon the proper delivery, efficient transcription, and optimal activity of sgRNA and Cas9 on intended target site and minimum unwanted genome perturbations, known as off-target effects. In this chapter, we will present a brief introduction and history of CRISPR/Cas followed by description of various tools used for its delivery and overview of gRNA design tools. Different resources are described which are used to validate the intended genome editing. Finally, we will conclude with future directions and the broader impact of CRISPR technologies.
Received in revised 08.08.2020 Methods based on the analysis of proteins and DNA molecules are more and more used to assess the composition of food products. Proteins research methods include immunological, electrophoretic and chromatographic ones. The analysis of DNA molecules is most often used to identify the species affiliation of food components. This is due to the stability of their structure compared to proteins, as well as their presence in most biological tissues. The results of studies evaluating methodological approaches for the application of the PCR diagnostic method to identify the composition of food products and the possibility of their use for monitoring dairy products have been shown. The objects of research were samples of cow, goat, sheep milk, as well as milk samples of different animal species mixed in various ratios. DNA was extracted from milk samples according to a unified technique for the separation of DNA molecules in milk and dairy products. The work also considers the possibility of using the PCR diagnostic method to identify the raw material origin of the product. To evaluate the measurement methods, artificially created samples of raw milk were used, which were cow, goat and sheep milk, a mix of three types of milk in different ratios. As a result of the research, the main method has been chosen as the real time PCR method, which has reliability, high sensitivity, sufficient rapidity, with the possibility of using it for dairy multicomponent products with a complex structural matrix, as well as products that have undergone deep technological processing.
Detection of genetically modified organisms (GMOs) in crops is an important issue for all the subjects involved in seed quality control and customer’s right. Due to the increasing number of GMOs research and development activity in the globe during the past few years, it has become necessary to screen and regulate highely practiced GMO crops. In this study, following the extraction of genomic DNA from agriculture farming land and commercial areas collected samples that suspected for GMOs such as Soybean and maize crops, the recombinant DNA target sequences were targeted to detect for transgene such as CaMV35s gene using thermal cycler Polymerase Chain Reaction (PCR). In addition, for positive control taxone specfic Invertase and Lectine gene of maize and soybean have done. Based on the gel electrophoresis results, Invertase and Lectine gene events were detected in all maize and soybean samples respectively. How ever, fortunately in all collected samples of maize and soybean in different places of the countery trans-genes were not detected. Finally, this reserch result concludes that in the studied area soybean and maize crops are GMO free.
The production and consumption of genetically modified (GM) products are highly controversial due to their environmental, health, and ethical impacts. Most of these disputes are caused by distrust of regulatory authorities, scientists, and technocratic decisions. Among all these concerns, health issues, allergenicity and antibiotic resistance are more important. Many of today's social development problems, including public health, energy, water scarcity, and economic problems are associated with the nutrition system. So, this article provides useful solutions for appropriate utilization of GM products at present and future and aims to improve the people's attitudes towards the impacts of biotechnology on food and agriculture. The current study was carried out in electronic databases, including Google Scholar, Pubmed, Scopus, Science direct, SID and Civilica using the following keywords: Genetically modified plants, GMOs safety, and Health risks. Information about biotechnology, transgenic products, technical methods for the assessment of these products, the organizations involved, and the benefits and disadvantages of GM products were recorded. Safe use of GM products could be achieved by approving specific rules in biotechnology, while respecting the safety principles, continuous evaluation and dissemination of information, and upgrading the public knowledge about these products.
The Enzyme-linked immunosorbent assay (ELISA) techniques employing highly sensitive and specific forms of immunological reactions find wide application in food analysis. The versatility in the functions of ELISA techniques render them suitable to detect specific constituents in food, including the naturally-occurring components, pesticide, therapeutic agents, beneficial and spoilage microorganisms, and toxins. It is a convenient and reliable analysis tool for the detection and quantification of constituents related to food production and processing, as well as food safety. The post-production of food products requires proper authenticity testing to ensure that their labeling do not falsify their adulterations. ELISA is also suitable in validation of such food adulterations thereby providing the consumers the benefit of making informed diet choices. The mostly used ELISA techniques in the food industry include indirect, sandwich and competitive ELISA that use both polyclonal and monoclonal antibodies as per the necessity. ELISA provides a suitable complementary approach in food analysis minimizing the use of sophisticated, expensive time-consuming techniques, yet maintaining the sensitivity and reliability of the outcome. Thus the present chapter aims to put forth the multitude of applicability of ELISA techniques in food analysis that have significant contributions in the food industry in terms of quality control and safety.
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.
An event-specific PCR method for detection and quantification of genetically modified Roundup Ready soybean (RRS) is described in this article. The complete DNA sequence at both the right and left integration sites of this genetically modified organism has recently been determined. Based on these sequence data, transformation event-specific primer pairs were developed. These primers amplify a fragment of the unique junction region between the inserted DNA and the plant DNA and therefore act as unique identifiers. Two sensitive, qualitative PCR assays gave absolute detection limits of 5 copies of the RRS junction fragment in 100 pg of total DNA per reaction. A real-time PCR method was then developed with the LightCycler System. For determination of the RRS content, a completely new type of external calibration standard is introduced here. A fragment of the RRS specific junction region and a fragment of the endogenous soybean lectin gene were both cloned in a plasmid vector. These new diagnostic DNA fragments allow quantification of RRS in whichever type of matrix, in a range of 10-106 copies of each target.
Numerous analytical methods, both qualitative and quantitative, have been developed to determine reliably the presence and/or the amount of genetically modified organisms (GMOs) in agricultural commodities, in raw agricultural materials and in processed and refined ingredients. In addition to the "classical" methods for DNA and protein analysis, e.g. polymerase chain reaction and enzyme linked immunosorbent analysis, certain types of GMO-containing matrices can be profiled by complementary chemical analysis methods such as chromatography and near infrared spectroscopy. This review summarises the status of the most widely used GMO analysis technologies, identifies new areas of analytical investigation and discusses current needs and future challenges.
An event-specific real-time PCR method for detection and quantification of genetically modified Roundup Ready soybean with TaqMan chemistry on the LightCycler, targeting the nopaline synthase terminator (3') junction between recombinant and host plant DNA is described. We distinguish between three types of detection and quantification limits: the absolute limits (referring to the initial number of template copies in the PCR), the relative limits (referring to the relative percentage of initial template copies of the recombinant sequence to copies of the haploid soybean genome that is detected), and the practical limits (referring to what is applicable in the PCR with the DNA that is being analysed). The absolute detection limit was determined to be a single initial template copy, while the absolute quantification limit was determined to be approximately 30 initial template copies. We discuss the relative and practical limits, and provide guidelines to estimating the practical limits.
In this article we describe the isolation and characterisation of the junction between insert DNA and plant DNA in the transgenic Roundup Ready soybean line event 40-3-2. Our results establish that during integration of the insert DNA several rearrangements occurred at the 3' NOS junction and that the genomic plant DNA at the pre-integration site may have been rearranged. These findings highlight the utility of characterising junction regions to fulfil the request for information regarding which DNA sequences have been incorporated in commercialised transgenic lines. Furthermore, the characterisation of junction regions is, in our opinion, the method of choice to support method development for detection and identification of plant biotechnology-derived products.
China has made a major investment in biotechnology research. Genetically modified (GM) cotton is widely adopted and the list of GM technologies in trials is impressive. At the same time there is an active debate on when China should commercialize its GM food crops. The overall goal of this paper is to provide an economy-wide assessment of these issues under various scenarios. Based on a unique data from empirical micro-level study and field trial in China and a modified GTAP model, our results indicate that the development of biotechnology has an important impact on China's production, trade and welfare. Welfare gains far outweigh the public biotechnology research expenditures. Most gains occur inside China. Policy makers should put less weight on the international dimension in making their decisions on biotechnology development.
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.
The presence of ingredients derived from genetically modified organisms (GMOs) in food products in the market place is subject to a number of European regulations that stipulate which product consisting of or containing GMO-derived ingredients should be labeled as such. In order to maintain these labeling requirements, a variety of different GMO detection methods have been developed to screen for either the presence of DNA or protein derived from (approved) GM varieties. Recent incidents where unapproved GM varieties entered the European market show that more powerful GMO detection and identification methods will be needed to maintain European labeling requirements in an adequate, efficient, and cost-effective way. This report discusses the current state-of-the-art as well as future developments in GMO detection.
Modification of genes through homologous recombination, termed gene targeting, is the most direct method to characterize gene function. In higher plants, however, the method is far from a common practice. Here we describe an efficient and reproducible procedure with a strong positive/negative selection for gene targeting in rice, which feeds more than half of the world's population and is an important model plant. About 1% of selected calli and their regenerated fertile plants were heterozygous at the targeted locus, and only one copy of the selective marker used was found at the targeted site in their genomes. The procedure's applicability to other genes will make it feasible to obtain various gene-targeted lines of rice.
New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye-labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0% level, the bias (mean-true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.
Although PCR technology has obvious limitations, the potentially high degree of sensitivity and specificity explains why it has been the first choice of most analytical laboratories interested in detection of genetically modified (GM) organisms (GMOs) and derived materials. Because the products that laboratories receive for analysis are often processed and refined, the quality and quantity of target analyte (e.g. protein or DNA) frequently challenges the sensitivity of any detection method. Among the currently available methods, PCR methods are generally accepted as the most sensitive and reliable methods for detection of GM-derived material in routine applications. The choice of target sequence motif is the single most important factor controlling the specificity of the PCR method. The target sequence is normally a part of the modified gene construct, for example a promoter, a terminator, a gene, or a junction between two of these elements. However, the elements may originate from wildtype organisms, they may be present in more than one GMO, and their copy number may also vary from one GMO to another. They may even be combined in a similar way in more than one GMO. Thus, the choice of method should fit the purpose. Recent developments include event-specific methods, particularly useful for identification and quantification of GM content. Thresholds for labelling are now in place in many countries including those in the European Union. The success of the labelling schemes is dependent upon the efficiency with which GM-derived material can be detected. We will present an overview of currently available PCR methods for screening and quantification of GM-derived DNA, and discuss their applicability and limitations. In addition, we will discuss some of the major challenges related to determination of the limits of detection (LOD) and quantification (LOQ), and to validation of methods.
We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5' 'HEAD' region and a 3' region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1-2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5' nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.
this report do not represent those of DG Research
This book is based on papers presented at the 4th meeting of the International Consortium on Agricultural Biotechnology Research on the 'Economics of Agricultural Biotechnology'. A subset of those papers is included in this volume, which addresses market development issues in developed countries, primarily in Europe and North America. Organized in 4 parts, this volume focuses on consumer reactions to GM foods, farmer acceptance of biotechnology products, the role of information systems and associated regulatory developments; and changes in industrial organization in life science and food sectors. It is aimed at those working in agricultural biotechnology and agricultural economics. The book has 26 chapters and a subject index.
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.
One of the most important breakthroughs in the history of genetics was the discovery that mutations can be induced (Muller, 1930; Stadler, 1932). The high frequency with which ionizing radiation and certain chemicals can cause genes to mutate made it possible to perform genetic studies that were not feasible when only spontaneous mutations were available.....
A method for analyzing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional
flatbed scanner is described here. Labeling oligonucleotide targets with nanoparticle rather than fluorophore probes substantially
alters the melting profiles of the targets from an array substrate. This difference permits the discrimination of an oligonucleotide
sequence from targets with single nucleotide mismatches with a selectivity that is over three times that observed for fluorophore-labeled
targets. In addition, when coupled with a signal amplification method based on nanoparticle-promoted reduction of silver(I),
the sensitivity of this scanometric array detection system exceeds that of the analogous fluorophore system by two orders
of magnitude.
Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA.
A piezoelectric affinity sensor, based on DNA hybridisation has been studied for applications to Genetically Modified Organisms (GMOs) detection. The thiol/dextran modified surfaces were coupled to streptavidin for immobilising 5'-biotinyltead probes (25-mer). The probes sequences were respectively internal to the amplified product of P35S and T-NOS. These target sequences were chosen on the base of their wide presence in GMOs. The system has been optimised using synthetic complementary oligonucleotides (25-mer) and the specificity of the system tested with a non-complementary oligonucleotide (23-mer). The hybridisation study was performed also with samples of DNA isolated from CRM (Certified Reference Materials) soybean powder containing 2% of transgenic material and amplified by PCR. Non amplified genomic or plasmidic DNA was also used. The developed system was very specific, binding only the complementary DNA strand. The CV% was 20% both with synthetic oligonucleotides and PCR amplified samples. The sensor signal was independent of the sample dilution but the system is still at a semi-quantitative level.
Ongoing technological advances permit the development of new scientific strategies for bio-analysis. The work described here sought to compare a traditional agarose slab gel electrophoresis method combined with digital image analysis against a new microfluidic capillary electrophoresis system (LabChipTM, Agilent Technologies) to assess their relative performance in the quantitative analysis of polymerase chain reaction (PCR) products. The comparative strategy exploited methodology for providing quantitative genetically modified organism (GMO) determinations in food samples and demonstrated that the LabChipTM system offered improvements in quantitation accuracy, objectivity and ease of use.
Demand for identity-preserved (IP) crops produced by Northern Plains farmers is increasing. Buyers are willing to pay a premium for grains that can be guaranteed to possess a unique characteristic. Several general crop management practices apply to crops raised for IP. These include greater investment in segregated storage facilities, more meticulous production, isolation, added cleaning/sorting, documentation, greater testing, additional marketing, and risks of liability. To illustrate, the economics of producing certified seed for sale to other farmers is used as an example of IP grain production. Many of the concepts and specific practices of certified seed production are applicable to most IP crops raised.
This study focuses on the economics of segregating U.S. non-biotech corn and soybeans for shipments to Japan, the primary export market for U.S. non-biotech grains and oilseeds, as a case study. The purposes of this paper are two-fold: 1) to estimate price premiums that buyers in both the U.S. domestic and Japanese export markets were willing to pay for non-biotech corn and soybeans for the 2000-2002 crops; and 2) to examine who bears the cost of segregation.
The expression of two cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3), has been investigated in MLA-144 cells before and after induction with phorbol 12-myristate 13-acetate. We describe an adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells. Aliquots of the PCR mixture containing cDNA copies of the RNA to be assayed were added to serial dilutions of a competitor DNA fragment that differed from the cDNA of interest by having either a small intron or a mutated internal restriction enzyme site. Therefore, the same primers were used to coamplify the unknown and the competitor. The ratio of products remains constant through the amplification and can be readily quantitated. In unstimulated cells, no GM-CSF or IL-3 mRNA could be detected. However, with appropriate induction, mRNA for both cytokines was detected and quantitated in as few as 200 cells. Competitive PCR was also used to accurately quantitate the copy number of the human GM-CSF gene in normal human cells, in a clonal population of cells from a patient with 5q- syndrome, and in a human-hamster cell line known to have only one copy of the human GM-CSF gene.
The factor V G1691A Leiden (FVL) mutation is the most common known hereditary risk factor for venous thrombosis.
Third Wave Technologies, Inc. (Madison, WI) has developed a new microtiter plate-based assay that does not require PCR, restriction digestion, or gel electrophoresis. This technology system, termed the Invader(TM) assay, utilizes a 5' "invading" oligonucleotide and a partially overlapping 3' "signal" oligonucleotide, which together form a specific structure when bound to a complementary genomic DNA template. A thermostable flap endonuclease cleaves this structure, releasing the 5' flap from the signal oligonucleotide. Increased temperature and an excess of the signal probe enable multiple probes to be cleaved for each target sequence present without temperature cycling. The cleaved probes then direct cleavage of a secondary probe, which is 5' end-labeled with fluorescein but is quenched by an internal dye. Upon cleavage, the fluorescein-labeled product is detected using a standard fluorescence plate reader. Genotypes are determined by net wild-type/mutant signal ratio.
Complete concordance was observed, after resolution of four discordances, when 1369 individuals (1264 wild type, 102 heterozygous, 3 homozygous) were FVL genotyped by both the Invader assay and by allele-specific PCR.
We conclude that FVL genotyping using invasive cleavage of oligonucleotide probes is a rapid and reliable alternative to genotyping by more traditional PCR-based methods.
The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.
A method for analyzing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional flatbed scanner is described here. Labeling oligonucleotide targets with nanoparticle rather than fluorophore probes substantially alters the melting profiles of the targets from an array substrate. This difference permits the discrimination of an oligonucleotide sequence from targets with single nucleotide mismatches with a selectivity that is over three times that observed for fluorophore-labeled targets. In addition, when coupled with a signal amplification method based on nanoparticle-promoted reduction of silver(I), the sensitivity of this scanometric array detection system exceeds that of the analogous fluorophore system by two orders of magnitude.
Homogeneity testing and the determination of minimum sample mass are an important part of the certification of reference materials. The smallest theoretically achievable uncertainty of certified concentration values is limited by the concentration distribution of analyte in the different particle size fractions of powdered biological samples. This might be of special importance if the reference material is prepared by dry mixing, a dilution technique which is used for the production of the new and third generation of genetically modified (GMO) plant certified reference materials. For the production of dry mixed PMON 810 maize reference material a computer program was developed to calculate the theoretically smallest uncertainty for a selected sample intake. This model was used to compare three differently milled maize samples, and the effect of dilution on the uncertainty of the DNA content of GMO maize was estimated as well. In the case of a 50-mg sample mass the lowest achievable standard deviation was 2% for the sample containing 0.1% GMO and the minimum deviation was less than 0.5% for the sample containing 5% GMO.
Suspension arrays of microspheres analyzed using flow cytometry offer a new approach to multiplexed assays for large-scale screening applications. By optically encoding micron-sized polymer particles, suspension microarrays can be created to enable highly multiplexed analysis of complex samples. Each element in the array is comprised of a subpopulation of particles with distinct optical properties and each array element bears a different surface receptor. Nucleic acids, proteins, lipids or carbohydrates can serve as receptors to support the analysis of a wide range of biomolecular assemblies, and applications in genomic and proteomic research are being developed. Coupled with recent innovations for rapid serial analysis of samples, molecular analysis with microsphere arrays holds significant potential as a general analysis platform for both research and clinical applications.
Biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect genetically modified Roundup Ready soybean gene sequences. We first immobilized, on SA sensor chips, single-stranded biotinylated oligonucleotides containing soybean lectin and Roundup Ready gene sequences, and the efficiency of hybridization to oligonucleotide probes differing in length was determined. Second, we immobilized biotinylated PCR products from nontransgenic soybeans (genomes carrying only the lectin gene), as well as from genetically modified Roundup Ready soybean, and we injected the oligonucleotide probes. Furthermore, we used the sensor chips carrying either lectin and Roundup Ready soybean PCR products or 21-mer oligonucleotide as probes, and we injected both nonpurified and purified asymmetric PCR products. The results obtained show that 13 and 15 mer oligonucleotides are suitable probes to detect genetically modified Roundup Ready soybean gene sequences (either target oligonucleotides or PCR products) under standard BIA experimental conditions. By contrast, when 11 mer DNA probes were employed, no efficient hybridization was obtained. All the SPR-based formats were found to be useful for detection of Roundup Ready gene sequences, suggesting that these procedures are useful for the real-time monitoring of hybridization between target single-stranded PCR products, obtained by using as substrates DNA isolated from normal or transgenic soybeans, and oligonucleotide or PCR-generated probes, therefore enabling a one-step, nonradioactive protocol to perform detection.
EU regulations stipulate the labeling of food products containing genetically modified organisms (GMOs) unless the GMO content is due to adventitious and unintended 'contamination' and not exceeding the 1% level at ingredient basis. In addition, member states have to ensure full traceability at all stages of the placing on the market of GMOs. Both requirements ensure consumers 'right to know', facilitate enforcement of regulatory requirements and are of importance for environmental monitoring and postmarket surveillance. Besides administrative procedures, such as used in quality certification systems, the significance of adequate molecular methods becomes more and more apparent. During the last decade a considerable number of molecular methods have been developed and validated that enable the detection, identification and quantification of GMO impurities. Most of them rely on the PCR technology and can only detect one specific stretch of DNA. It can, however, be anticipated that in the near future the situation will become more complex. The number of GMO varieties, including 'stacked-gene' varieties, which will enter the European Market will increase and it is likely that these varieties will harbor more variable constructs. New tools will be necessary to keep up with these developments. One of the most promising techniques is microarray analysis. This technique enables the screening for a large number of different GMOs within a single experiment.
Multiplexed detection of oligonucleotide targets has been performed with gold nanoparticle probes labeled with oligonucleotides
and Raman-active dyes. The gold nanoparticles facilitate the formation of a silver coating that acts as a surface-enhanced
Raman scattering promoter for the dye-labeled particles that have been captured by target molecules and an underlying chip
in microarray format. The strategy provides the high-sensitivity and high-selectivity attributes of gray-scale scanometric
detection but adds multiplexing and ratioing capabilities because a very large number of probes can be designed based on the
concept of using a Raman tag as a narrow-band spectroscopic fingerprint. Six dissimilar DNA targets with six Raman-labeled
nanoparticle probes were distinguished, as well as two RNA targets with single nucleotide polymorphisms. The current unoptimized
detection limit of this method is 20 femtomolar.
Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material requires pure, high-quality genomic DNA as template for subsequent amplification using the polymerase chain reaction (PCR). Six methodologies were evaluated for extracting DNA from ground corn kernels spiked with 0.1% (m/m) CBH351 (StarLink) corn. DNA preparations were evaluated for purity and fragment size. Extraction efficiency was determined. The alcohol dehydrogenase gene (adh1) and the CBH351 (cry9C, 35S promoter) genes in the genomic DNA were detected using PCR. DNA isolated by two of the methods proved unsuitable for performing PCR amplification. All other methods produced some DNA preparations that gave false negative PCR results. We observed that cornstarch, a primary component of corn kernels, was not an inhibitor of PCR, while acidic polysaccharides were. Our data suggest that amplification of an endogenous positive control gene, as an indicator for the absence of PCR inhibitors, is not always valid. This study points out aspects of DNA isolation that need to be considered when choosing a method for a particular plant/tissue type.
This paper aims to examine some of the reasons behind public controversy associated with the introduction of genetically modified foods in Europe the 1990s. The historical background to the controversy is provided to give context. The issue of public acceptance of genetically modified foods, and indeed the emerging biosciences more generally, is considered in the context of risk perceptions and attitudes, public trust in regulatory institutions, scientists, and industry, and the need to develop communication strategies that explicitly include public concerns rather than exclude them. Increased public participation has been promoted as a way of increasing trust in institutional practices associated with the biosciences, although questions still arise as to how to best utilise the outputs of such exercises in policy development. This issue will become more of a priority as decision-making systems become more transparent and open to public scrutiny. The results are discussed in the context of risk assessment and risk management, and recommendations for future research are made. In particular, it is recommended that new methods are developed in order to integrate public values more efficaciously into risk analysis processes, specifically with respect to the biosciences and to technology implementation in general.
The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment. This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding. The document then focuses on the potential value of evolving "profiling" or "omics" technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined.
This paper provides guidance on how to assess the safety of foods derived from genetically modified crops (GM crops); it summarises conclusions and recommendations of Working Group 1 of the ENTRANSFOOD project. The paper provides an approach for adapting the test strategy to the characteristics of the modified crop and the introduced trait, and assessing potential unintended effects from the genetic modification. The proposed approach to safety assessment starts with the comparison of the new GM crop with a traditional counterpart that is generally accepted as safe based on a history of human food use (the concept of substantial equivalence). This case-focused approach ensures that foods derived from GM crops that have passed this extensive test-regime are as safe and nutritious as currently consumed plant-derived foods. The approach is suitable for current and future GM crops with more complex modifications. First, the paper reviews test methods developed for the risk assessment of chemicals, including food additives and pesticides, discussing which of these methods are suitable for the assessment of recombinant proteins and whole foods. Second, the paper presents a systematic approach to combine test methods for the safety assessment of foods derived from a specific GM crop. Third, the paper provides an overview on developments in this area that may prove of use in the safety assessment of GM crops, and recommendations for research priorities. It is concluded that the combination of existing test methods provides a sound test-regime to assess the safety of GM crops. Advances in our understanding of molecular biology, biochemistry, and nutrition may in future allow further improvement of test methods that will over time render the safety assessment of foods even more effective and informative.
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