Applying and developing DNA-based methods for detecting genetically modified plants

Testing for GM plant materials in dockage

Some importing countries have concerns about the possible presence of unapproved or discontinued GM events in dockage materials found in wheat and barley cargo shipments. In a recent study, we looked for the presence of unapproved GM events in dockage materials collected from wheat and barley grain shipments. The most frequent type of dockage material found in barley and wheat shipments was canola seed. We tested canola dockage samples for the presence of different GM events using polymerase chain reaction (PCR) and pre-spotted plates. Pre-spotted plates use aliquots of primers and probes specific for each GM event. This method allowed us to detect 11 different GM events in canola at concentrations as low as 0.01% (Table 1). The most commonly detected GM canola events were GT73, MS8 and RF3. These GM events have received regulatory approval in the European Union and many other countries and pose no regulatory issues.

We analyzed 1,038 samples of dockage material and did not detect the unauthorized OXY235 and the discontinued HCN92 GM canola events. Only trace levels (≤0.1%) of GM events RF1 (3 samples), MS1 (3 samples), T45 (2 samples) and RF2 (1 sample) were found. These discontinued GM events had approval in the EU but are now being phased out. Overall, the risk of unapproved or discontinued GM canola events being found in dockage materials collected from barely and wheat shipments was found to be very low.

Increasing the efficiency of GM event detection

We use droplet digital PCR (ddPCR) technology to measure the absolute amount of a GM event in a target sample without comparing it to a reference sample. The target sample is divided into thousands of partitions or droplets and each droplet is tested for the presence of a given GM event. Usually, only one GM event is detected with a ddPCR assay and if we need to measure more than one GM event in a sample, the assay is performed multiple times. We investigated the feasibility of absolute quantification of two, three and four GM events at the same time using the QX200 Droplet Digital PCR system. To successfully use multiplex ddPCR, an adjustment of probe concentrations and labels was needed for some assays. For probe optimization, all four GM events were run individually using FAM and HEX probe dyes in triplicates to determine the signal intensity and establish the cluster location. Based on the results, the probes for the GM events to be labeled with either FAM or HEX were determined. Then, all four GM events were run together to assess the pattern and separation of clusters (Figure 1).

We achieved the absolute quantification of GM events in canola and soybean using duplex, triplex and tetraplex ddPCR assays at concentrations of 0.1%, 1% and 5%. The multiplex ddPCR will allow us to detect and quantify GM events more efficiently, reducing the time and resources required for monitoring.

Recent monitoring activities

Between August 1, 2021, and July 31, 2022, we analyzed the variety composition of the following:

• 380 samples from wheat cargo
• 135 samples from durum cargo
• 62 samples from monthly cargo composites
• 64 samples from wheat co-op trials
• 36 wheat samples from the Harvest Sample Program
• 14 submitted barley cargo samples

In addition, 419 wheat cargo samples were analyzed for the presence of MON71200 GM event. Bulk testing of wheat cargo samples for the presence of this GM event began in March 2018 and was discontinued on April 1, 2022. In all the samples we analyzed, MON71200 was never detected. The test is still available for submitted samples on a fee-for-service basis.