Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR.

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.

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.

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

The 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 abstract There 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 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.

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.

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.

Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.

The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain.

A multiplex PCR procedure was established to detect Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain. The PCR protocol with an enrichment step successfully detected all three organisms inoculated together in non-autoclaved wheat grain. After a one day enrichment, E. coli, L. monocytogenes and S. typhimurium were detected at levels of 56, 1800 and <54 CFU/mL, respectively, in the initial sample. For L. monocytogenes, an improved detection limit of <62 CFU/mL was achieved using singleplex PCR. For autoclaved wheat grain inoculated with the three bacterial strains individually, a detection limit of 3 CFU/mL was achieved after an enrichment step. The ability to test for the three bacteria simultaneously will save time and increase the ability to assure grain quality.

Species-specific PCR-based assays for the detection of Fusarium species and a comparison with the whole seed agar plate method and trichothecene analysis.

Species-specific PCR was used for the identification of nine Fusarium species in pure mycelial culture. A PCR-based method was compared with the whole seed agar plate method and trichothecene analysis for three toxin-producing Fusarium species using 85 grain samples of wheat, barley, oat, corn and rye. A simple SDS-based DNA extraction system followed by potassium acetate precipitation resulted in consistent PCR amplification of DNA fragments from cultures and grain samples. The species-specific PCR assays correctly identified pure cultures of Fusarium avenaceum ssp. avenaceum (9 isolates), Fusarium acuminatum ssp. acuminatum (12 isolates), Fusarium crookwellense (7 isolates), Fusarium culmorum (12 isolates), Fusarium equiseti (11 isolates), Fusarium graminearum (77 isolates), Fusarium poae (10 isolates), Fusarium pseudograminearum (23 isolates), and Fusarium sporotrichioides (10 isolates). Multiplex PCR was developed for the simultaneous detection of F. culmorum, F. graminearum and F. sporotrichioides, the three most important trichothecene producing species in Canada. In grain samples, results of PCR assays for these same three species related well with whole seed agar plate method results and determination of Fusarium trichothecenes. The PCR assay described in this study can be used for routine detection and identification of Fusarium spp. in Canada.