Detection Of Transgenic DNA Research Articles

Health status and potential uptake of transgenic DNA by Japanese quail fed diets containing genetically modified plant ingredients over 10 generations

The hypothesis assumes that feed containing GMOs affects animal health and results in the transgene product accumulating in the body. Therefore, the objective of the study was to evaluate the impact of genetically modified (GM) ingredients used in poultry diets on aspects of bird health status and accumulation of transgenic DNA in eggs, breast muscle and internal organs.A total of 10 generations of Japanese quail were fed three types of diets: group A – containing GM soya (Roundup Ready) and non-GM maize, group B – containing GM maize (MON810) and non-GM soya, and group C – containing non-GM soya and maize.Bird performance traits were monitored throughout the trial. In 17-week-old animals of each generation, health examination took place on birds from each group including post-mortem necropsy and histological organ evaluation. For the purpose of transgenic DNA detection, samples of selected important tissues were taken. A molecular screening method of PCR amplification was used.The analysis of the sectional examination of birds used in the current experiment did not indicate the existence of the pathological changes caused by pathogens, nutritional factors or of environmental nature. The histopathological changes occurred in all three dietary groups and there were no statistically significant differences between the groups.There was no transgene amplification – neither CaMV35S promoter sequence nor nos terminator sequence, in the samples derived from breast muscle, selected tissues and germinal discs (eggs).According to the obtained results, it was concluded that there was no negative effect of the use of GM soya or maize with regard to bird health status or to the presence of transgenic DNA in the final consumable product.

Detection of EPO gene doping in blood

Gene doping--or the abuse of gene therapy--will continue to threaten the sports world. History has shown that progress in medical research is likely to be abused in order to enhance human performance. In this review, we critically discuss the progress and the risks associated with the field of erythropoietin (EPO) gene therapy and its applicability to EPO gene doping. We present typical vector systems that are employed in ex vivo and in vivo gene therapy trials. Due to associated risks, gene doping is not a feasible alternative to conventional EPO or blood doping at this time. Nevertheless, it is well described that about half of the elite athlete population is in principle willing to risk its health to gain a competitive advantage. This includes the use of technologies that lack safety approval. Sophisticated detection approaches are a prerequisite for prevention of unapproved and uncontrolled use of gene therapy technology. In this review, we present current detection approaches for EPO gene doping, with a focus on blood-based direct and indirect approaches. Gene doping is detectable in principle, and recent DNA-based detection strategies enable long-term detection of transgenic DNA (tDNA) following in vivo gene transfer.

Simultaneous Detection of Transgenic DNA by Surface Plasmon Resonance Imaging with Potential Application to Gene Doping Detection

Surface plasmon resonance imaging (SPRi) was used as the transduction principle for the development of optical-based sensing for transgenes detection in human cell lines. The objective was to develop a multianalyte, label-free, and real-time approach for DNA sequences that are identified as markers of transgenosis events. The strategy exploits SPRi sensing to detect the transgenic event by targeting selected marker sequences, which are present on shuttle vector backbone used to carry out the transfection of human embryonic kidney (HEK) cell lines. Here, we identified DNA sequences belonging to the Cytomegalovirus promoter and the Enhanced Green Fluorescent Protein gene. System development is discussed in terms of probe efficiency and influence of secondary structures on biorecognition reaction on sensor; moreover, optimization of PCR samples pretreatment was carried out to allow hybridization on biosensor, together with an approach to increase SPRi signals by in situ mass enhancement. Real-time PCR was also employed as reference technique for marker sequences detection on human HEK cells. We can foresee that the developed system may have potential applications in the field of antidoping research focused on the so-called gene doping.

A PCR assay to detect trace amounts of soybean in meat sausages

SummarySoybean proteins are the most widely used source vegetable proteins in the meat industry because of several interesting characteristics. As soybean is included in the group of ingredients potentially allergenic, if not declared, it can be considered a hidden allergen, representing a potential risk to sensitised individuals. The aim of this work was to optimise and apply DNA‐based techniques for soybean detection in meat products, as alternative to the currently used protein‐based methods. The optimised polymerase chain reaction (PCR) protocol targeting the soybean lectin gene enabled the detection of the addition of 0.1% and 0.5% of hydrated textured protein, which corresponded to 0.01% and 0.06% (w/w) of soybean protein in unprocessed and heat‐processed pork meats, respectively. The established PCR technique, when applied to commercial meat sausages (eighteen samples), confirmed the presence of soybean declared in nine samples and indicated the presence of soybean in four samples with no labelled information about soybean. Additionally, the event‐specific PCR detection of Roundup Ready® soybean was also performed, enabling the detection of transgenic DNA in three samples.

A simple DNA extraction method suitable for PCR detection of genetically modified maize

The polymerase chain reaction (PCR) is a powerful tool that is being increasingly used for detection of transgenic DNA. PCR requires only a minute quantity of template, but sensitive and accurate testing requires DNA of sufficient purity and free from inhibitors such as plant polysaccharides. Several standard protocols are available for this purpose, but they usually involve several steps, imply destruction of the maize kernel, or are time-consuming. Our aim was to develop a fast and simple extraction method to isolate a raw DNA-containing solution from maize tissues suitable for use as a template in a PCR-based detection assay with specific oligonucleotides directed to the identification of event MON810. The NaOH-based DNA extraction method we report here is time-saving (5 min) and can be used to isolate DNA-containing solutions from a small maize leaf portion (down to 1 mg) or from a single overnight-germinated kernel. PCR performed with selected primers yielded reproducible detection of transgenic DNA. The main advantages of the procedure are the quick extraction step, the possibility of non-destructive testing of maize kernels, and the robustness of the PCR-based detection, a consequence of the selection of MON810-matching oligonucleotides yielding intense and highly specific amplicons. Copyright © 2007 Society of Chemical Industry.

Detection of Genetically Modified Crops and Their Derivatives: Critical Steps in Sample Preparation and Extraction

The detection of genetically modified crops in foodstuff relies on detection of transgenic DNA or protein material in the sample matrix. Purified DNA or proteins are used as analytical material for polymerase chain reaction technologies and immunodiagnostics. Successful sample preparation is critical to the validity of subsequent analysis. For routine analysis, a good sample preparation technique should be simple, safe, and inexpensive while reproducibly generating DNA/protein of sufficient quality and yield. The suitability of isolated DNA or protein as an analyte for a detection or characterization technique depends on amount or concentration, purity, and integrity, each of which may be influenced by sample matrix and the extraction technique, and, in turn, may impact the validity of analytical techniques. The key sample preparation steps of homogenization, pretreatment, extraction, and purification are discussed as well as typical analytical methods. Consideration is given to application of these steps for particular sample matrixes to maximize yield, reduce inhibition effects, and minimize contamination. The choice of the most appropriate and valid methods for sample preparation from particular foods is discussed with respect to DNA analysis. Attention is also given to ease of use, cost, and generic applicability of the procedures.

Detection of transgenic DNA in milk from cows receiving herbicide tolerant (CP4EPSPS) soyabean meal

Ten Holstein/Friesian cows with an average liveweight of 630 kg and producing 25.3 kg milk/day received a total mixed ration (TMR) in which the forage component formed 55% of total DM and contained non-genetically modified (GM) grass and maize silage in a 1:3 DM ratio. In study weeks 1–3 the TMR DM also contained non-GM supplements of 18.5% cracked wheat and 26.1% rapeseed meal. In weeks 4–12 soyabean meal, genetically modified (CP4EPSPS) to be herbicide tolerant, replaced rapeseed meal at 26.1% of the total diet in weeks 4–5 and 13.9% of the total diet in weeks 6–12. Weekly milk samples were taken from all cows. Samples were spiked with DNA extracted from the soyabean meal to establish the limit of detection (LOD) of transgenic DNA using a polymerase chain reaction (PCR) analyses designed to detect transgenic DNA fragments smaller than 200 bp. Subsequent PCR analyses carried out in duplicate on TMR and milk samples collected at weeks 3, 4 and 12 of the study used three DNA primer sets to establish the presence or absence of transgenic DNA. The LOD of transgenic DNS in milk was 7.5 μg/l. Feed and milk samples analysed at week 3 were negative. The TMR feed samples at weeks 4 and 12 were positive for CP4EPSPS soyabean meal DNA, but all milk samples were negative. The results showed that transgenic DNA could not be detected in milk from cows receiving upto 26.1% of their diet DM as herbicide (glyphosate)-tolerant soyabean meal.

Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico.

Concerns have been raised about the potential effects of transgenic introductions on the genetic diversity of crop landraces and wild relatives in areas of crop origin and diversification, as this diversity is considered essential for global food security. Direct effects on non-target species, and the possibility of unintentionally transferring traits of ecological relevance onto landraces and wild relatives have also been sources of concern. The degree of genetic connectivity between industrial crops and their progenitors in landraces and wild relatives is a principal determinant of the evolutionary history of crops and agroecosystems throughout the world. Recent introductions of transgenic DNA constructs into agricultural fields provide unique markers to measure such connectivity. For these reasons, the detection of transgenic DNA in crop landraces is of critical importance. Here we report the presence of introgressed transgenic DNA constructs in native maize landraces grown in remote mountains in Oaxaca, Mexico, part of the Mesoamerican centre of origin and diversification of this crop.