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1D-Amplitude plots of a ddPCR PCR assay applied on the p87701 plasmid containing 1000 copies each of the transgene MON 87701 and the reference gene lectin. Samples were analyzed in triplicates containing varying proportions of SDS as PCR-inhibitor.
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Droplet digital polymerase chain reaction (ddPCR) has seen increasing applications in recent times, also in the analysis of genetically modified (GM) food and feed samples. While quantitative real-time PCR (qPCR) methods have been traditional mainstays till now, the applicability of ddPCR in routine analysis of GM food and feed has not yet been wid...
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Context 1
... the SDS concentrations tested, ddPCR appeared to be more stable in performance, compared to the real-time qPCR assay (Fig. 2, panel 3). The SDS concentrations tested were in the range of 0.001 % -0.01 % (end concentration), with the SDS-solution, directly added to the master mix in place of water (see ...
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... include a subset of partially inhibited samples (sometimes referred to as "rain"), while a higher threshold, placed close to the clustering of positive droplets would be more appropriate for uninhibited samples (Dingle, et al., 2013). Interestingly, the degree of sensitivity to SDS of transgene and reference gene showed a slight asymmetry (Fig. 3), with more rain (intermediate signals between clearly positive and negative signals) visible with the transgene in the FAM channel, compared with the reference gene lectin in ...
Citations
... In contrast to the target gene, ZmLOX5, an almost equal copy number concentration was determined in each individual for the reference gene, α-Tubulin, representing the single copy gene. The relative copy numbers of ZmLOX5 were calculated using α-Tubulin as internal controls [33,35], and the results reveal that only B73 contained a one-fold change in the ZmLOX5/α-Tubulin ratio, while Yu796 and the three independent B73-ZmLOX5 lines contained two-fold changes in ZmLOX5/α-Tubulin ratios, indicating that these individuals carry duplicate copies of ZmLOX5 whereas B73 encodes a single copy of ZmLOX5, as expected ( Figure 1C). ...
Extensive genome structure variations, such as copy number variations (CNVs) and presence/absence variations, are the basis for the remarkable genetic diversity of maize; however, the effect of CNVs on maize herbivory defense remains largely underexplored. Here, we report that the naturally occurring duplication of the maize 9-lipoxygenase gene ZmLOX5 leads to increased resistance of maize to herbivory by fall armyworms (FAWs). Previously, we showed that ZmLOX5-derived oxylipins are required for defense against chewing insect herbivores and identified several inbred lines, including Yu796, that contained duplicated CNVs of ZmLOX5, referred to as Yu796-2×LOX5. To test whether introgression of the Yu796-2×LOX5 locus into a herbivore-susceptible B73 background that contains a single ZmLOX5 gene is a feasible approach to increase resistance, we generated a series of near-isogenic lines that contained either two, one, or zero copies of the Yu796-2×LOX5 locus in the B73 background via six backcrosses (BC6). Droplet digital PCR (ddPCR) confirmed the successful introgression of the Yu796-2×LOX5 locus in B73. The resulting B73-2×LOX5 inbred line displayed increased resistance against FAW, associated with increased expression of ZmLOX5, increased wound-induced production of its primary oxylipin product, the α-ketol, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (9,10-KODA), and the downstream defense hormones regulated by this molecule, 12-oxo-phytodienoic acid (12-OPDA) and abscisic acid (ABA). Surprisingly, wound-induced JA-Ile production was not increased in B73-2×LOX5, resulting from the increased JA catabolism. Furthermore, B73-2×LOX5 displayed reduced water loss in response to drought stress, likely due to increased ABA and 12-OPDA content. Taken together, this study revealed that the duplicated CNV of ZmLOX5 quantitively contributes to maize antiherbivore defense and presents proof-of-concept evidence that the introgression of naturally occurring duplicated CNVs of a defensive gene into productive but susceptible crop varieties is a feasible breeding approach for enhancing plant resistance to herbivory and tolerance to abiotic stress.
... In contrast to the target gene, ZmLOX5, almost equal copy number concentration was determined in each individual for the reference gene, α-Tubulin, representing the single copy gene. The relative copy numbers of ZmLOX5 were calculated using α-Tubulin as internal control [26,32], and the results revealed that only B73 contained one-fold change in the ZmLOX5/α-Tubulin ratio, while Yu796 and the three independent B73-ZmLOX5 lines contained two-fold changes in ZmLOX5/α-Tubulin ratios, indicating these individuals carry duplicate copies of ZmLOX5, whereas B73 encodes a single copy of ZmLOX5, as expected ( Figure 1C). ...
Extensive genome structure variation, such as copy number variations (CNVs) and presence/absence variations, is the basis for the remarkable maize genetic diversity; however, the effect of CNVs on maize herbivory defense remains largely underexplored. Here, we report that the naturally occurring duplication of the maize 9-lipoxygenase gene, ZmLOX5, leads to increased resistance of maize to herbivory by fall armyworms (FAW). Previously, we showed that ZmLOX5-derived oxylipins are required for defense against chewing insect herbivores and identified several inbred lines, including Yu796, that contained duplicated CNVs of ZmLOX5, referred to as Yu796-2×LOX5. To test whether introgression of the Yu796-2×LOX5 locus into the herbivore-susceptible B73 background that contains a single ZmLOX5 gene is a feasible approach to increase resistance, we generated a series of near-isogenic lines that contained either 2, 1 or 0 copies of the Yu796-2×LOX5 locus in the B73 background by six back crosses (BC6). Droplet digital PCR (ddPCR) confirmed the successful introgression of the Yu796-2×LOX5 locus in B73. The resulting B73-2×LOX5 inbred line displayed increased resistance against FAW associated with increased expression of ZmLOX5, increased wound-induced production of its primary oxylipin product, the α-ketol, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (9,10-KODA), and the downstream defense hormones regulated by this molecule, 12-oxo-phytodienoic acid (12-OPDA) and abscisic acid (ABA). Surprisingly, wound-induced JA-Ile production was not increased in B73-2×LOX5, resulting from the increased JA catabolism. Furthermore, B73-2×LOX5 displayed enhanced drought tolerance likely due to increased ABA and 12-OPDA content. Taken together, this study revealed that the duplicated CNV of ZmLOX5 quantitively contributes to maize antiherbivore defense and presents proof-of-concept evidence that the introgression of naturally occurring duplicated CNVs of a defensive gene into the productive, but susceptible, crop varieties is a feasible breeding approach for enhancing plant resistance to herbivory and tolerance to abiotic stress.
... robust multiplex quantification. This is particularly important for complex samples where qPCR-based quantification is difficult due to sensitivity to inhibitors often present in GMO samples [5][6][7][8][9][10]. ...
The proliferation of genetically modified organisms (GMOs) presents challenges to GMO testing laboratories and policymakers. Traditional methods, like quantitative real-time PCR (qPCR), face limitations in quantifying the increasing number of GMOs in a single sample. Digital PCR (dPCR), specifically multiplexing, offers a solution by enabling simultaneous quantification of multiple GMO targets. This study explores the use of the Naica six-color Crystal dPCR platform for quantifying five GM soybean lines within a single six-plex assay. Two four-color assays were also developed for added flexibility. These assays demonstrated high specificity, sensitivity (limit of detection or LOD < 25 copies per reaction) and precision (bias to an estimated copy number concentration <15%). Additionally, two approaches for the optimization of data analysis were implemented. By applying a limit-of-blank (LOB) correction, the limit of quantification (LOQ) and LOD could be more precisely determined. Pooling of reactions additionally lowered the LOD, with a two- to eight-fold increase in sensitivity. Real-life samples from routine testing were used to confirm the assays’ applicability for quantifying GM soybean lines in complex samples. This study showcases the potential of the six-color Crystal dPCR platform to revolutionize GMO testing, facilitating comprehensive analysis of GMOs in complex samples.
... In contrast, several issues with Southern hybridization can cause problems with resolution (Sun and Joyce, 2017), a problem experienced in the present study with the use of non-radioactive methodology. The ddPCR method has proven effective with several crops other species, including Brassica (Demeke and Eng, 2018), maize (Collier et al. 2017) and soybean (Iwobi et al. 2016), as cited by Giraldo et al. (2019), often in the context of testing feed and food for transgenic material. ...
A reliable protocol for Agrobacterium-mediated genetic transformation of Lens culinaris Medik (lentil) was developed. Using cultivar Laird, the protocol yielded rooted shoots from an average of 6.8 independent events per hundred seeds. The protocol utilized longitudinal slices of embryo axes from imbibed mature seed as a starting explant and a plasmid containing a β-glucuronidase:neomycin phosphotransferase (gus:nptII) fusion gene in Agrobacterium strain EHA105. A series of four media, each with appropriate levels of kanamycin selection were identified and other factors tested included the optical density of the Agrobacterium suspension, and type and concentration of plant growth regulators. The expression of the gus reporter gene was visualized through histochemical staining, and further molecular analysis through PCR, qPCR, ddPCR and Southern hybridization confirmed transformation and provided copy number. The inserted genes were inherited into the T1 generation and chimaeras were not identified. The time from co-cultivation to the planting of rooted shoots ranged from 4 to 7 months, as transgenic clusters continue to produce additional clonal shoots.
... Secara keseluruhan, kombinasi suhu ruang dan waktu inkubasi semalam menunjukkan kuantitas dan kualitas DNA yang paling optimal pada kegiatan ekstraksi DNA C. intybus (Tabel 7). Hasil ini senada dengan penelitian sebelumnya yang dilakukan oleh Paithankar (Iwobi et al. 2016). ...
DNA extraction is a crucial stage in plant molecular analysis activities that determine the success of further downstream analysis stages. Molecular analysis activities, including Polymerase Chain Reaction (PCR), restriction digestion, DNA hybridisation, genomic library construction, and DNA sequencing, mostly require DNA with high quality and enough quantity. Some plants contain high levels of polysaccharides, proteins, and secondary metabolites, such as polyphenols, tannins, and alkaloids, which can affect the purity of the isolated DNA. The presence of these contaminants can inhibit the activity of several enzymes, such as DNA polymerase and restriction enzymes, so that DNA could not be amplified or digested. Various DNA extraction methods have been developed, ranging from manual method, manual commercial kits, and robotic
techniques. Each method has its advantages and disadvantages since the selection of the right method needs to be
determined carefully in order to obtain the best results. Some modifications at certain stages in DNA extraction process need to be conducted to minimize the number of DNA contaminants. Several rapid DNA extraction methods nowadays have also been developed to save times, labors, and costs. In addition, the rapid DNA extraction method could also reduce the use of
some hazardous chemical compounds, so that the method is not only giving economic benefits, but also good for human health.
Keywords: DNA, extraction, plant molecular analysis, genomic.
... EDTA-2Na, however, is also known to be a PCR inhibitor. 36,37 It is thus important to know if EDTA-2Na could be used to release the trapped viruses by dissolving the aluminum hydroxide precipitates without sacrificing the subsequent PCR test performance. ...
This study aimed to provide a low-cost technique for virus detection in wastewater by improving an aluminum hydroxide adsorption-precipitation method. The releasing efficiency of viruses trapped by the aluminum hydroxide precipitates was improved by adding ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) to dissolve the precipitates at a Na2EDTA·2H2O:AlCl3 molar ratio of 1.8-3.6. The recovery rates of the improved method for seven viruses, including SARS-CoV-2-abEN pseudovirus and six animal viruses, were 5.9-22.3% in tap water and 4.9-35.1% in wastewater. Rotavirus A (9.0-4.5 × 103 copies/mL), porcine circovirus type 2 (5.8-6.4 × 105 copies/mL), and porcine parvovirus (5.6-2.7 × 104 copies/mL) were detected in China's pig farm wastewater, while rotavirus A (2.0 × 103 copies/mL) was detected in hospital wastewater. SARS-CoV-2 was detected in hospital wastewater (8.4 × 102 to 1.4 × 104 copies/mL), sewage (6.4 × 10 to 2.3 × 103 copies/mL), and river water (6.6 × 10 to 9.3 × 10 copies/mL) in Nepal. The method was automized, with a rate of recovery of 4.8 ± 1.4% at a virus concentration of 102 copies/mL. Thus, the established method could be used for wastewater-based epidemiology with sufficient sensitivity in coping with the COVID-19 epidemic and other virus epidemics.
... Moreover, this end-point PCR technology is less sensitive to inhibitors due to the partitioning of the sample into thousands of droplets by a water-oil emulsion. The measurement uncertainty is thus reduced, in particular at low target copy number (Cottenet et al., 2019;Dobnik et al., 2016;Dobnik, Spilsberg, Košir, Holst-Jensen, & Ž el, 2015;Gerdes et al., 2016;Grelewska-Nowotko et al., 2018;Iwobi et al., 2016;Kosir et al., 2017;Kosir et al., 2019;Morisset et al., 2013;Wan et al., 2016). ...
Gene-edited organisms and derived food and feed products commercialized on the European market falls within the scope of the Directive, 2001/18/EC. Therefore, the possibility to specifically detect and quantify them has become a priority. To this end, PCR-based approaches, such as real-time PCR and digital droplet PCR, targeting a single variation point carried by a gene-edited organism are expected to be suitable, even if potentially challenging at the technical level. However, additional issues related to the interpretation of the results can also be encountered. Indeed, given its possible spread, natural or through breeding programs, the presence of this single variation does not automatically prove the presence of the gene-edited organism. To overcome such critical issue, we proposed a general workflow to develop and validate a PCR-based method specific to a gene-edited organism in targeting its single variation point. First, based on in silico analyses, the possibility to technically design the PCR-based method as well as to discriminate the gene-edited organism using it single variation point are assessed. In case such parameters are confirmed, the performance of the developed PCR-based method are then tested in agreement with the minimum performance requirements for GMO testing. The use of the proposed general workflow was successfully illustrated through the development a 2-plex digital droplet PCR method targeting specifically a gene-edited rice carrying a single nucleotide insertion. The proposed workflow was thus considered as a key tool to support the competent authorities regarding the food and feed traceability.
... An inter-laboratory dPCR study comprising seven independent laboratories showed less than 4.5% reproducibility relative standard deviation for a high GC-rich reference material [13]. dPCR assays have also been reported to be less sensitive to inhibitors in the DNA samples compared with real-time quantitative PCR assays [15][16][17]. However, ethanol has been reported to affect the stability of droplets for dPCR [15]. ...
... dPCR assays have also been reported to be less sensitive to inhibitors in the DNA samples compared with real-time quantitative PCR assays [15][16][17]. However, ethanol has been reported to affect the stability of droplets for dPCR [15]. ...
The number of genetically modified (GM) events for canola, maize, and soybean has been steadily increasing. Real-time PCR is widely used for the detection and quantification of individual GM events. Digital PCR (dPCR) has also been used for absolute quantification of GM events. A duplex dPCR assay consisting of one reference gene and one GM event has been carried out in most cases. The detection of more than one GM event in a single assay will increase the efficiency of dPCR. The feasibility of detection and quantification of two, three, and four GM canola and soybean events at the same time was investigated at 0.1%, 1%, and 5% levels using the QX200 Droplet Digital PCR (ddPCR) system. The reference gene assay was carried out on the same plate but in different wells. For some of the assays, optimization of the probe concentrations and labels was needed for successful ddPCR. Results close to the expected result were achieved for duplex, triplex, and tetraplex ddPCR assays for GM canola events. Similar ddPCR results were also achieved for some GM soybean events with some exceptions. Overall, absolute quantification of up to four GM events at the same time improves the efficiency of GM detection.
... Thus, an accurate and precise CN measurement is critical for lines selection, being single or low CN generally desired [38]. Many studies have been carried out to compare the performances of qPCR and ddPCR in view of improving detection and quantification methods intended for those laboratories committed in official GMO control [15,[39][40][41][42][43][44]. In general, they argued in favor to ddPCR, being this technique insensitive to PCR inhibiting components often present in complex matrices and not dependent on calibration with standard curves obtained from certified reference materials for quantification. ...
Agrobacterium tumefaciens- mediated gene transfer—actually the most used method to engineer plants—may lead to integration of multiple copies of T-DNA in the plant genome, as well as to chimeric tissues composed of modified cells and wild type cells. A molecular characterization of the transformed lines is thus a good practice to select the best ones for further investigation. Nowadays, several quantitative and semi-quantitative techniques are available to estimate the copy number (CN) of the T-DNA in genetically modified plants. In this study, we compared three methods based on (1) real-time polymerase chain reaction (qPCR), (2) droplet digital PCR (ddPCR), and (3) next generation sequencing (NGS), to carry out a molecular characterization of grapevine edited lines. These lines contain a knock-out mutation, obtained via CRISPR/Cas9 technology, in genes involved in plant susceptibility to two important mildew diseases of grapevine. According to our results, qPCR and ddPCR outputs are largely in agreement in terms of accuracy, especially for low CN values, while ddPCR resulted more precise than qPCR. With regard to the NGS analysis, the CNs detected with this method were often not consistent with those calculated by qPCR and ddPCR, and NGS was not able to discriminate the integration points in three out of ten lines. Nevertheless, the NGS method can positively identify T-DNA truncations or the presence of tandem/inverted repeats, providing distinct and relevant information about the transgene integration asset. Moreover, the expression analysis of Cas9 and single guide RNA (sgRNA), and the sequencing of the target site added new information to be related to CN data. This work, by reporting a practical case-study on grapevine edited lines, explores pros and cons of the most advanced diagnostic techniques available for the precocious selection of the proper transgenic material. The results may be of interest both to scientists developing new transgenic lines, and to laboratories in charge of GMO control.
... Soybean (Glycine max L. Merr.) accounts for 78% for worldwide individual crop region (94.1 million hectares), and the maize (Zea mays L.) for 33% (59.7 million hectares), and the cotton (Gossypium arboretum L.) for 80% (24.21 million hectares), and canola (Brassica napus L.) for 30% (Brassica napus L.) (10.2 million hectares). Sugar beet (Beta vulgaris L.), papaya (Carica papaya L.), squash (Cucurbita L.), potatoes (Solanum tuberosum L.), and apples (Maluspumila Miller, 1768) have all recently been added to the list of transgenes that can be used commercially is-delivered crops cultivars, and these products are now commercially available in United States [62,63]. Similarly the organic compound or the secondary metabolites such as drugs or chemicals are produced by entophytic mildew. ...
Regulatory bodies around the world are scheduled to begin performing tests on genetically modified animals as developer's plant modifiers that move from the experimental to the commercial phase of their research. Necessary safety evaluation products of food are derived from these animals. Considerable experience in the developments of these anticipants has accumulated in food industry during the last three decades. It is being used to examine the safety of genetically modified plants and microbes. Design new animal-derived product criteria that takes into account the Foods which are made from plants, and those made from animals have certain similarities as well as some variance sources. Therefore we want powerful structures to evaluate GMO's (genetically modified organizations) in feed stuffs from the dietary factor of view. The most significant condition for public adoption to obtain feed and food is safe and nutritional studies with such transformed plants. New crop varieties with altered genetic elements, on the other hand, may be submitted to safety studies before marketing to meet regulatory criteria. The main goal of this review is to determine the influence of novel crops and their products on animal, environment and health. While several related studies on the change of assessment for genetically modified food had been published, and little attention has been paid to genetically modify feed stuff, even though animal feed accounts for 70% and 90% of all crops having the amount of living matter. The study discussed scheme for nutritional assessment of GMOs, including the search for unanticipated consequences components of safety assessment.
