Event-specific Assay Research Articles

An integrated strategy involving high-throughput sequencing to characterize an unknown GM wheat event in Canada.

Glyphosate-resistant wheat plants were discovered in southern Alberta in 2017, representing an unauthorized GM release in Canada. The Canadian Food Inspection Agency undertook a series of experiments to characterize and identify this unknown GM wheat, as well as to develop and validate construct-specific and event-specific qPCR assays. Results of PCR-based assays and Sanger sequencing indicated the presence of CaMV 35S promoter (p35S), Rice Actin 1 intron (RactInt1), CP4-EPSPS gene and nopaline synthase terminator (tNOS) elements in the unknown GM wheat. Genome walking and bead capture strategies, combined with high-throughput sequencing, were used to identify the 5' and 3' wheat junctions and the subsequent mapping of the insert to chromosome 3B of the wheat genome. A probable transformation vector, pMON25497, was recognized, and further testing identified the unknown GM wheat as MON71200 event, one of two events obtained with the pMON25497 vector. The two construct-specific assays targeted the junctions of the RactInt1 and the CP4-EPSPS elements and the CP4-EPSPS and tNOS elements, while the event-specific assay was located at the 3' junction into the wheat genome. Both construct-specific and event-specific assays had limits of detection of 0.10% of MON71200 in a seed pool. As expected, the two construct-specific assays cross-reacted with other wheat and corn events containing the same elements in the same order. No cross-reactivity was observed for the event-specific assay. The integrated strategy employed in this study can serve as a model for other cases when facing similar challenges involving unknown GM events.

An event-specific method for the detection and quantification of ML01, a genetically modified Saccharomyces cerevisiae wine strain, using quantitative PCR

The availability of genetically modified (GM) yeasts for winemaking and, in particular, transgenic strains based on the integration of genetic constructs deriving from other organisms into the genome of Saccharomyces cerevisiae, has been a reality for several years. Despite this, their use is only authorized in a few countries and limited to two strains: ML01, able to convert malic acid into lactic acid during alcoholic fermentation, and ECMo01 suitable for reducing the risk of carbamate production. In this work we propose a quali-quantitative culture-independent method for the detection of GM yeast ML01 in commercial preparations of ADY (Active Dry Yeast) consisting of efficient extraction of DNA and qPCR (quantitative PCR) analysis based on event-specific assay targeting MLC (malolactic cassette), and a taxon-specific S. cerevisiae assay detecting the MRP2 gene. The ADY DNA extraction methodology has been shown to provide good purity DNA suitable for subsequent qPCR. The MLC and MRP2 qPCR assay showed characteristics of specificity, dynamic range, limit of quantification (LOQ) limit of detection (LOD), precision and trueness, which were fully compliant with international reference guidelines. The method has been shown to reliably detect 0.005% (mass/mass) of GM ML01 S. cerevisiae in commercial preparations of ADY.

PCR-Based Detection and Quantification of a Transgenic Glyphosate-Tolerant Canola Using a Novel Reference Gene System

Development and commercialization of a transgenic crop depends upon the availability of a reliable quantitative assay sensitive enough to detect low level presence of transgenic material in mixed seed or in a commercial product. An appropriate species-specific endogenous control also is necessary for accurate quantification. A novel mode of transgenic tolerance to glyphosate, whereupon an engineered acetylase detoxifies glyphosate, was recently developed and incorporated into transgenic canola (Brassica napus). Here, we describe two highly specific and sensitive PCR-based assays for the transgenic event, DP-O73496-4. We also developed and evaluated an appropriate endogenous control to be used in conjunction with the event-specific assays. This endogenous control is specific to the A genome of cultivated canola-quality oilseed rape based on our analysis of the diversity of FatA gene sequences.

International ring trial for the validation of an event-specific Golden Rice 2 quantitative real-time polymerase chain reaction method.

This article describes the international validation of the quantitative real-time polymerase chain reaction (PCR) detection method for Golden Rice 2. The method consists of a taxon-specific assay amplifying a fragment of rice Phospholipase D α2 gene, and an event-specific assay designed on the 3' junction between transgenic insert and plant DNA. We validated the two assays independently, with absolute quantification, and in combination, with relative quantification, on DNA samples prepared in haploid genome equivalents. We assessed trueness, precision, efficiency, and linearity of the two assays, and the results demonstrate that both the assays independently assessed and the entire method fulfill European and international requirements for methods for genetically modified organism (GMO) testing, within the dynamic range tested. The homogeneity of the results of the collaborative trial between Europe and Asia is a good indicator of the robustness of the method.

Genetics, structure, and prevalence of FP967 (CDC Triffid) T-DNA in flax.

The detection of T-DNA from a genetically modified flaxseed line (FP967, formally CDC Triffid) in a shipment of Canadian flaxseed exported to Europe resulted in a large decrease in the amount of flax planted in Canada. The Canadian flaxseed industry undertook major changes to ensure the removal of FP967 from the supply chain. This study aimed to resolve the genetics and structure of the FP967 transfer DNA (T-DNA).The FP967 T-DNA is thought to be inserted in at single genomic locus. The junction between the T-DNA and genomic DNA consisted of two inverted Right Borders with no Left Border (LB) flanking genomic DNA sequences recovered. This information was used to develop an event-specific quantitative PCR (qPCR) assay. This assay and an existing assay specific to the T-DNA construct were used to determine the genetics and prevalence of the FP967 T-DNA. These data supported the hypothesis that the T-DNA is present at a single location in the genome.The FP967 T-DNA is present at a low level (between 0.01 and 0.1%) in breeder seed lots from 2009 and 2010. None of the 11,000 and 16,000 lines selected for advancement through the Flax Breeding Program in 2010 and 2011, respectively, tested positive for the FP967 T-DNA, however.Most of the FP967 T-DNA sequence was resolved via PCR cloning and next generation sequencing. A 3,720 bp duplication of an internal portion of the T-DNA (including a Right Border) was discovered between the flanking genomic DNA and the LB. An event-specific assay, SAT2-LB, was developed for the junction between this repeat and the LB.

Event-specific analytical methods for six genetically modified maize events using visual and real-time loop-mediated isothermal amplification

Currently 138 genetically modified (GM) maize events have been authorized for commercial cultivation, comprising more than 65 per cent stacked events. With the increase in number of GM maize events globally, cost- and time-efficient diagnostics with on-site applicability are required to check for authorized GM events. Six GM maize events, namely, Bt11, GA21, MON810, MON89034, NK603 and TC1507, also present in 89 stacked events, are being widely commercialized in more than 17 countries. Visual and real-time loop-mediated isothermal amplification (LAMP) assays targeting these six GM maize events are being reported in the present study. Specificity of the developed LAMP assays was confirmed using fourteen commercialized GM maize events. Limit of detection of visual and real-time LAMP assays targeting Bt11, GA21, MON810, MON89034 and TC1507, was up to 0.01%, detecting 8 target copies, and for NK603 event-specific assays, was up to 0.1% detecting 73 target copies. Practical applicability of developed LAMP assays was verified using a set of five stacked GM maize events, namely, Bt11 × GA21, MON89034 × NK603, MON89034 × NK603 × TC1507, TC1507 × NK603 and TC1507 × MON810; and six powdered maize samples of proficiency testing. The reported LAMP assays can be efficiently employed for screening for presence of selected GM maize events in single or stacked form.

Development of Phaseolus vulgaris endogenous reference and Embrapa 5.1 event-specific assays for quantification of Embrapa 5.1 GM common bean using real time PCR

Background Brazil is the second largest producer of genetically modified (GM) crops with 37 GM crop varieties approved by CTNBio for commercialization. One of these varieties is the Embrapa 5.1 common bean, resistant to the bean golden mosaic virus (BGMV). The Embrapa 5.1 event was developed using the RNAi concept to induce the silencing of the AC1 viral gene [1]. Labelling of food products containing GMOs in Brazil is mandatory [2], so methods for quantification of GMOs are required. Real time PCR methods have been used as the gold standard for GMO quantification. In a previous work, we have developed a construct-specific PCR detection assay for Embrapa 5.1 [3]. In this study we developed an event-specific PCR assay for Embrapa 5.1.

Development of an event-specific assay for the qualitative and quantitative detection of the genetically modified flax CDC Triffid (FP967)

The genetically modified flax, event FP967, with tolerance to sulfonylurea herbicides, is one of the commercial genetically modified events approved in Canada and USA.This event is not authorized in Switzerland and EU, therefore, a method to specifically detect the CDC Triffid line, was required. We revealed the 3′ integration junction sequence between host plant DNA and the integrated gene construct FP967 by means of Restriction Site PCR and both a qualitative and a quantitative PCR detection assays were developed. The qualitative PCR revealed a limit of detection of 0.01% of GM flax in 100 ng of genomic DNA. The quantitative PCR assay showed a limit of detection of about 9 haploid genome copies. The specificity and sensitivity of the assay indicate that the developed event-specific PCR methods can be used for identification and quantification of FP967 flax.

Multiplex Real-Time PCR Kit for Qualitative Detection of Genetically Modified Roundup Ready Soybean

A multiplex real-time polymerase chain reaction (PCR) kit was developed for the qualitative detection of the Roundup-Ready Soybean (RRS). This system consists of a screening assay for cauliflower mosaic virus 35S promoter and nos terminator, and an event-specific assay for RRS containing the lectin gene as an internal positive control. The limit of detection value was determined to be approximately 0.1%.

Event-Specific Qualitative and Quantitative PCR Detection Methods for Transgenic Rapeseed Hybrids MS1×RF1 and MS1×RF2

Except for the events RT73, MS8, RF3, and T45, event-specific detection methods for most commercialized genetically modified (GM) rapeseed varieties have not been established, and as a result, the enforcement of genetically modified organism labeling policies has been hindered. The genetically modified rapeseeds, MS1xRF1 and MS1xRF2, are 2 of 11 approved GM-rapeseed varieties for commercialization. In this study, the right border junction fragments between the gene construct and the rapeseed genome of events RF1, RF2, and MS1 were isolated using the commercially available GenomeWalker technology. Homology analysis indicated that the gene construct of RF1 integrated upstream of the nuclease gene, and that of the RF2 and MS1 inserted into the exon region of a gene encoding for an unknown protein. The event-specific primer pairs and corresponding probes were designed on the basis of the revealed right border junction fragments. Then, we successfully developed the identification and quantification methods for the gene-stacked hybrids MS1xRF1 and MS1xRF2 using those primers and probes. The relative limit of detection in the qualitative polymerase chain reaction (PCR) was 0.013% for the RF2 and MS1 assays using 100 ng of rapeseed DNA per reaction and 0.13% for the RF1 assay. The absolute limit of detection in the quantitative PCR was approximately one to two initial copies for each of the three event-specific assays. The evaluation of the real-time PCR assays revealed that the qualitative and quantitative methods developed by focusing on the gene-stacked hybrids MS1xRF1 and MS1xRF2 were highly specific, sensitive, and suitable for samples with a low quantity of DNA.

Comparison of Real-Time PCR Detection Chemistries and Cycling Modes Using Mon810 Event-Specific Assays as Model

The most widely accepted methods for accurate quantitative detection of genetically modified organisms rely on real-time PCR. Various detection chemistries are available for real-time PCR. They include sequence-unspecific DNA labeling dyes such SYBR-Green I and the use of both universal (e.g., AmpliFluor) and sequence-specific double-labeled probes, the latter comprising hybridization (e.g., Molecular Beacon) and hydrolysis (e.g., TaqMan or MGB) probes. Also, new real-time PCR devices and reagents allowing fast cycling reactions exist. Five Mon810 real-time PCR assays were developed in which the event specificity was based on the detection of transgene and plant rearranged sequences found to 3' flank the insertion site. Every assay was specifically designed for one particular detection chemistry, that is, AmpliFluor, Molecular Beacon, MGB, TaqMan, and SYBR-Green I. When possible, the assays were adapted to fast cycling mode. All assays displayed satisfactory performance parameters, although Molecular Beacon, MGB, and TaqMan chemistries were the most suitable for quantification purposes in both conventional and fast cycling modes.

Event-specific detection of Roundup Ready Soya using two different real time PCR detection chemistries

Recently, the DNA sequences of both the 5' and 3' sites of integration of the transgene in Roundup Ready soya have been described. We have utilised this information to design two event-specific assays for the quantitative detection of this genetically modified (GM) organism in food matrices using real time PCR. One assay has been designed to use TaqMan technology for detection using the ABI PRISM 7700 and the second to utilise Scorpion primers for use on the Roche Diagnostics LightCycler detection system. In both cases, the Roundup Ready gene was quantified relative to the endogenous soya lectin gene and results expressed as the percentage of GM soya relative to total soya content using standards of known GM content.