Tigst Demeke

Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits

Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.

Simultaneous profiling of seed‐associated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds

In order to address the hypothesis that seeds from ecologically and geographically diverse plants harbor characteristic epiphytic microbiota, we characterized the bacterial and fungal microbiota associated with Triticum and Brassica seed surfaces. The total microbial complement was determined by amplification and sequencing of a fragment of chaperonin 60 (cpn60). Specific microorganisms were quantified by qPCR. Bacteria and fungi corresponding to operational taxonomic units (OTU) that were identified in the sequencing study were isolated and their interactions examined. A total of 5477 OTU were observed from seed washes. Neither total epiphytic bacterial load nor community richness/evenness was significantly different between the seed types; 578 OTU were shared among all samples at a variety of abundances. Hierarchical clustering revealed that 203 were significantly different in abundance on Triticum seeds compared with Brassica. Microorganisms isolated from seeds showed 99–100% identity between the cpn60 sequences of the isolates and the OTU sequences from this shared microbiome. Bacterial strains identified as Pantoea agglomerans had antagonistic properties toward one of the fungal isolates (Alternaria sp.), providing a possible explanation for their reciprocal abundances on both Triticum and Brassica seeds. cpn60 enabled the simultaneous profiling of bacterial and fungal microbiota and revealed a core seed-associated microbiota shared between diverse plant genera.

Development of a specific TaqMan real-time PCR assay for quantification of Fusarium graminearum clade 7 and comparison of fungal biomass determined by PCR with deoxynivalenol content in wheat and barley.

A Fusarium graminearum clade 7 specific real-time quantitative PCR (qPCR) assay was developed in this study based on unique polymorphisms in sequences of the mating type protein (MAT) gene. PCR amplification was not observed in eight phylogenetic lineages of the F. graminearum complex and four other closely related Fusarium species. Accuracy of the quantification of the real-time PCR assay was verified with wheat DNA spiked with F. graminearum clade 7 DNA. Wheat samples representing two Canadian wheat classes, CWRS (Canadian Western Red Spring) and CWRW (Canadian Western Red Winter) were used to determine the relationships among F. graminearum DNA, deoxynivalenol (DON) and Fusarium damaged kernel (FDK). The amount of DON and F. graminearum DNA remaining after removal of FDK varied among samples, but was sometimes substantial. Positive correlations were observed between F. graminearum clade 7 DNA (in picograms) and DON as well as FDK. There was also a strong correlation between FDK and DON in CWRS and CWRW wheat composite samples, but the inherent variability in individual producer samples precluded a definitive correlation. For barley, a positive correlation was observed between Fusarium DNA and DON values. Real-time PCR assays can be a valuable tool for barley as there are no reliable symptoms to visually assess the level of Fusarium head blight in this crop.

Deoxynivalenol levels and chemotype frequency in barley cultivars inoculated with two chemotypes of Fusarium graminearum

Abstract Corn kernels colonized by Fusarium graminearum were spread between lines of barley (Hordeum vulgare) in an irrigated Fusarium head blight (FHB) nursery at a 2 : 1 ratio of isolates producing 15-acetyldeoxynivalenol (15 ADON) to isolates producing 3-acetyldeoxynivalenol (3 ADON) (in 2008) or 1 : 1 ratio (in 2009, 2010). In 2008, it was the first use in this field of a 3 ADON isolate after 9 years of artificial inoculation with exclusively 15 ADON isolates. Sets of three 2-row and three 6-row cultivars were used as checks throughout the nursery, representing a range of susceptibility to FHB. Composites of these cultivars were prepared and analysed for per cent F. graminearum infection, chemotype frequency and levels of deoxynivalenol (DON), 3 ADON and 15 ADON. Seed infection by F. graminearum ranged from 83.5% (2009) to 98.5% (2010), with little difference across the nursery. In 2008, the chemotype distribution of F. graminearum and DON levels on infected kernels showed a several-fold difference from the eastern side (67% 3 ADON isolates and 46 ppm DON) to the western end of the field (16% 3 ADON isolates and 13 ppm DON), but not in the subsequent 2 years. In both 2009 and 2010, the 3 ADON chemotype was recovered from only 18% and 13% of the seeds, and overall average DON levels were 18 ppm and 24 ppm, respectively. In 2008, the highest DON and 3 ADON levels were associated with the highest frequency of the 3 ADON chemotype. In all years, the recovery of isolates did not reflect the ratio of the initial inoculum, suggesting that other factors influenced the ultimate infection of the seed. However, the relative relationship among the barley cultivars was consistent throughout all 3 years: DON levels were much lower in the most resistant cultivars compared with the most susceptible cultivars in all years and composites. Genotypic response to DON accumulation did not show strong interaction with 3 ADON frequency, suggesting that the underlying genetic resistance of the barley cultivars is cross-applicable between chemotypes.

Effect of Source of DNA on the Quantitative Analysis of Genetically Engineered Traits Using Digital PCR and Real-Time PCR.

Seven commercially available DNA extraction kits were compared with a cetyltrimethylammonium bromide (CTAB) method to determine the suitability of the extracted DNA for RainDrop digital PCR (dPCR) and real-time PCR (RT-PCR) quantification of OXY235 canola, FP967 flax, and DP305423 soybean (spiked at the 0.1% level). For the kits, the highest amount of DNA extracted from a 0.2 g sample was obtained using OmniPrep for Plant for flax and DNeasy mericon Food for canola and soybean. For canola, DNA extracted with the Fast ID Genomic DNA Extraction Kit, FastDNA Spin Kit, GM Quicker 2, NucleoSpin Food, and DNeasy mericon Food was suitable for dPCR and RT-PCR. For flax, DNA extracted with Fast ID, FastDNA Spin Kit, OmniPrep for Plant, and NucleoSpin Food was suitable for RT-PCR. However, only Fast ID yielded DNA suitable for dPCR. For soybean, DNA extracted with five and six of the seven DNA extraction kits was suitable for dPCR and RT-PCR, respectively. Overall, Fast ID provided reliable results regardless of species or analysis method used. Canola, flax, and soybean DNA extracted with the CTAB method and then purified were suitable for both dPCR and RT-PCR. This is the first report showing the effect of different DNA extraction methods on the absolute quantification of genetically engineered traits using dPCR.

Influence of amount of starting material for DNA extraction on detection of low-level presence of genetically engineered traits.

Two laboratories independently examined how the amount of starting material influences DNA extraction efficiency and, ultimately, the detection of low-level presence of genetically engineered (GE) traits in commercialized grains. GE traits from one maize, two canola, and two soybean samples were used as prototypical models in the study design as well as two commonly used DNA extraction methods, a small scale (0.1 and 0.2 g samples) and a large scale (1.0 and 2.0 g samples). The DNA samples were fortified (spiked) at 0.1 and 0.01% (w/w) levels. The amount of DNA recovery varied between the two laboratories, although a sufficient amount of DNA was obtained to perform replicate PCR analysis by both laboratories. Reliable detection of all five events was achieved by both laboratories at 0.1% level using either small-scale or large-scale DNA extractions. Reliable detection of the GE events was achieved at 0.01% level for soybean and canola but not for maize. Variability was observed among the two laboratories in terms of the Ct values generated. There was no difference between small-scale and large-scale DNA extraction methods for qualitative PCR detections of all five GE events.

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

AbstractThe number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing. Graphical abstractThere are generally three different options for absolute quantification of genetically modified organisms (GMOs) using digital PCR: droplet- or chamber-based and droplets in chambers. All have in common the distribution of reaction mixture into several partitions, which are all subjected to PCR and scored at the end-point as positive or negative. Based on these results GMO content can be calculated.

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events

Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences (HMG-I/Y, FatA(A), CruA and Ccf) for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A), reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A) reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences) were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes.